Purification of vaccinia virus- and recombinant vaccinia virus-based vaccines

ABSTRACT

The present invention relates to methods for purification of Vaccinia viruses (VV) and/or Vaccinia virus (VV) particles, which can lead to highly pure and stable virus preparations of predominantly biologically active viruses. The invention encompasses purifying a virus preparation in a sterilized way with high efficiency and desirable yield in terms of purity, biological activity and stability, aspects advantageous for industrial production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/622,474, filed Nov. 20, 2009, now U.S. Pat. No. 8,003,363, which is acontinuation-in-part of U.S. application Ser. No. 12/598,362, filed Oct.30, 2009 (now U.S. Pat. No. 8,012,738), which is the U.S. National Stageof International Application No. PCT/EP2008/003679 filed May 7, 2008,which claims the benefit of U.S. Provisional Application No. 60/924,413,filed May 14, 2007, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods for purification of Vaccinia viruses(VV) and/or Vaccinia virus (VV) particles.

2. Description of Related Art

Traditionally in medicine, a vector is a living organism that does notcause disease itself, but which spreads infection by “carrying”pathogens (agents that cause disease) from one host to another. Avaccine vector is a weakened or killed version of a virus or bacteriumthat carries an inserted antigen (coding for a protein recognized by thebody as foreign) from a disease-causing agent to the subject beingvaccinated. A vaccine vector delivers the antigen in a natural way intothe body and stimulates the immune system into acting against a “safeinfection.” The immune system is led into generating an immune responseagainst the antigen that protects the vaccinated subject against future“risky infections.”

In vaccine development, a recombinant modified virus can be used as thevehicle or vaccine vector for delivering genetic material to a cell.Once in the cell, genetic information is transcribed and translated intoproteins, including the inserted antigen targeted against a specificdisease. Treatment is successful if the antigen delivered by the vectorinto the cell produces a protein, which induces the body's immuneresponse against the antigen and thereby protects against the disease.

A viral vector can be based on an attenuated virus, which cannotreplicate in the host but is able to introduce and express a foreigngene in the infected cell. The virus or the recombinant virus is therebyable to make a protein and display it to the immune system of the host.Some key features of viral vectors are that they can elicit a stronghumoral (B-cell) and cell-mediated (T-cell) immune response.

Viral vectors are commonly used by researchers to develop vaccines forthe prevention and treatment of infectious diseases and cancer, and ofthese, poxviruses (including canary pox, vaccinia, and fowl pox) are themost common vector vaccine candidates.

Pox viruses are a preferred choice for transfer of genetic material intonew hosts due to the relatively large size of the viral genome (appr.150/200 kb) and because of their ability to replicate in the infectedcell's cytoplasm instead of the nucleus, thereby minimizing the risk ofintegrating genetic material into the genome of the host cell. Of thepox viruses, the vaccinia and variola species are the two best known.The virions of pox viruses are large as compared to most other animalviruses (for more details see Fields et al., eds., Virology, 3^(rd)Edition, Volume 2, Chapter 83, pages 2637 if).

Variola virus is the cause of smallpox. In contrast to variola virus,vaccinia virus does not normally cause systemic disease inimmune-competent individuals and it has therefore been used as a livevaccine to immunize against smallpox. Successful worldwide vaccinationwith Vaccinia virus culminated in the eradication of smallpox as anatural disease in the 1980s (The global eradication of smallpox. Finalreport of the global commission for the certification of smallpoxeradication; History of Public Health, No. 4, Geneva: World HealthOrganization, 1980). Since then, vaccination has been discontinued formany years, except for people at high risk of poxvirus infections (forexample, laboratory workers). However, there is an increasing fear that,for example, variola causing smallpox may be used as a bio-terrorweapon. Furthermore, there is a risk that other poxviruses such ascowpox, camelpox, and monkeypox may potentially mutate, throughselection mechanisms, and obtain similar phenotypes as variola. Severalgovernments are therefore building up stockpiles of Vaccinia-basedvaccines to be used either pre-exposure (before encounter with variolavirus) or post-exposure (after encounter with variola virus) of apresumed or actual smallpox attack.

Vaccinia virus is highly immune-stimulating and provokes strong B-(humoral) and T-cell mediated immunity to both its own gene products andto any foreign gene product resulting from genes inserted in theVaccinia genome. Vaccinia virus is therefore seen as an ideal vector forvaccines against smallpox and other infectious diseases and cancer inthe form of recombinant vaccines. Most of the recombinant Vacciniaviruses described in the literature are based on the fully replicationcompetent Western Reserve strain of Vaccinia virus. It is known thatthis strain has a high neurovirulence and is thus poorly suited for usein humans and animals (Morita et al. 1987, Vaccine 5, 65-70).

In contrast, the Modified Vaccinia virus Ankara (MVA) is known to beexceptionally safe. MVA has been generated by long-term serial passagesof the Chorioallantois Vaccinia Ankara (CVA) strain of Vaccinia virus onchicken embryo fibroblast (CEF) cells (for review see Mayr, A. et al.1975, Infection 3, 6-14; Swiss Patent No. 568,392). Examples of MVAvirus strains deposited in compliance with the requirements of theBudapest Treaty are strains MVA 572, MVA 575, and MVA-BN® deposited atthe European Collection of Animal Cell Cultures (ECACC), Salisbury (UK)with the deposition numbers ECACC V94012707, ECACC V00120707 and ECACCV00083008, respectively, and described in U.S. Pat. Nos. 7,094,412 and7,189,536.

MVA is distinguished by its great attenuation profile compared to itsprecursor CVA. It has diminished virulence or infectiousness, whilemaintaining good immunogenicity. The MVA virus has been analyzed todetermine alterations in the genome relative to the wild type CVAstrain. Six major deletions of genomic DNA (deletion I, II, III, IV, V,and VI) totaling 31,000 base pairs have been identified (Meyer, H. etal. 1991, J. Gen. Virol. 72, 1031-1038). The resulting MVA virus becameseverely host-cell restricted to avian cells. The excellent propertiesof the MVA strain have been demonstrated in extensive clinical trials(Mayr, A. et al. 1978, Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390;Stickl, H. et al. 1974, Dtsch. med. Wschr. 99, 2386-2392), where MVA 571has been used as a priming vaccine at a low dose prior to theadministration of conventional smallpox vaccine in a two-step programand was without any significant adverse events (SAES) in more than120,000 primary vaccinees in Germany (Stickl, H et al. 1974, Dtsch. med.Wschr. 99, 2386-2392; Mayr et al. 1978, Zbl. Bakt. Hyg. I, Abt. Org. B167, 375-390).

MVA-BN® is a virus used in the manufacturing of a stand-alone thirdgeneration smallpox vaccine. MVA-BN® was developed by further passagesfrom MVA strain 571/572. To date, more than 1500 subjects includingsubjects with atopic dermatitis (AD) and HIV infection have beenvaccinated in clinical trials with MVA-BN® based vaccines.

The renewed interest in smallpox vaccine-campaigns with Vaccinia-basedvaccines has initiated an increased global demand for large-scalesmallpox vaccine production. Furthermore, the use of Vaccinia virus as atool for preparation of recombinant vaccines has additionally createdsignificant industrial interest in methods for manufacturing (growth andpurification) of native Vaccinia viruses and recombinant-modifiedVaccinia viruses.

Viruses used in the manufacturing of vaccines or for diagnostic purposescan be purified in several ways depending on the type of virus.Traditionally, purification of pox viruses including Vaccinia virusesand recombinant-modified Vaccinia viruses has been carried out based onmethods separating molecules by means of their size differences. Toenhance removal of host cell contaminants (e.g. DNA and proteins), inparticular DNA, the primary purification by means of size separation hasbeen supplemented by secondary methods such as enzymatic digestion ofDNA (e.g. Benzonase treatment). Most commonly, the primary purificationof Vaccinia viruses and recombinant-modified Vaccinia viruses has beenperformed by sucrose cushion or sucrose gradient centrifugation atvarious sucrose concentrations. Recently, ultrafiltration has also beenapplied either alone or in combination with sucrose cushion or sucrosegradient purification.

Vaccinia Viruses-based vaccines have in general been manufactured inprimary CEF (Chicken Embryo Fibroblasts) cultures. Vaccines manufacturedin primary CEF cultures are generally considered safe as regardsresidual contaminants. First, it is scientifically unlikely that primarycell cultures from healthy chicken embryos should contain any harmfulcontaminants (proteins, DNA). Second, millions of people have beenvaccinated with vaccines manufactured on CEF cultures without anyadverse effects resulting from the contaminants (CEF proteins and CEFDNA). There is, therefore, no regulatory requirement for the level ofhost cell contaminants in vaccines manufactured in primary CEF cultures,but for each vaccine the manufacturer must document its safety. Theregulatory concern for vaccines manufactured in primary CEF culturesrelates to the risk of adventitious agents (microorganisms (includingbacteria, fungi, mycoplasma/spiroplasma, mycobacteria, rickettsia,viruses, protozoa, parasites, TSE agent) that are inadvertentlyintroduced into the production of a biological product).

In the current methods for purification of Vaccinia viruses,manufactured in primary CEF culture the level of CEF protein may be upto 1 mg/dose and the CEF DNA level may exceed 10 μg/dose of 1×10⁸ asmeasured by the TCID50. These levels are considered acceptable from asafety and regulatory perspective as long as the individual vaccinemanufacturer demonstrates that the levels to be found in the Final DrugProduct (FDP) are safe at the intended human indications. Due to therisk of presence of adventitious agents in vaccines manufactured inprimary cell cultures and the associated need for extensive, expensivebiosafety testing of each vaccine batch manufactured, there is a strongstimulus for the vaccine industry to change to continuous cell lines.Once a continuous cell line has been characterized the need for testingfor adventitious agents of the production batches is minimal.

However, switch from primary to continuous cell culture for productionof Vaccinia and Vaccinia recombinant vaccines is expected to imposestricter safety and regulatory requirements. In fact, the regulatoryauthorities have proposed new requirements for levels of DNAcontaminants in vaccines manufactured using continuous cell lines (SeeDraft FDA guideline), which may be as low as 10 ng host-cell DNA/dose.To achieve such low level of host cell contaminants, new and improvedmethods for purification are needed.

It appears that vaccinia virions are able to bind to heparin through thesurface protein A27L (Chung et al. 1998, J. Virol. 72, 1577-1585). Atleast three surface proteins A27L (Chung et al., J. Virol.72(2):1577-1585, 1998; Ho et al., Journal of Molecular Biology349(5):1060-1071, 2005; Hsiao et al., J. Virol. 72(10):8374-8379, 1998)D8L (Hsiao et al., J. Virol. 73(10):8750-8761, 1999), and H3L (Lin etal., J. Virol. 74(7):3353-3365, 2000) of the most abundant infectiousform of the Vaccinia virus have been reported to bind toglycosaminoglycans.

It has further been suggested that affinity chromatography (Zahn, A andAllain, J.-P. 2005, J. Gen. Virol. 86, 677-685) may be used as basis forpurification of certain virus preparations. There are several examplesfor the application of ion exchange and affinity membrane adsorbers (MA)for the purification of virus particles like adenoviral vectors (Peixotoet al., Biotechnology Progress 24(6):1290-1296, 2008; Sellick, BioPharmInternational 19(1):31-32, 34, 2006), Aedes aegyptidensonucleosis virus(Enden et al., J Theor Biol 237(3):257-264, 2005), baculovirus (Wu etal., Hum. Gene Ther. 18(7):665-672, 2007), and influenza virus (Kalbfusset al., Journal of Membrane Science 299(1-2):251-260, 2007; Opitz etal., Biotechnol. and Bioeng. 103(6):1144-1154, 2009; and Opitz et al.,Journal of Biotechnology 131(3):309-317, 2007).

For efficient purification of vaccinia virus and recombinant vacciniavirus-based vaccines, some significant challenges need to be overcome.Vaccinia virions are far too large to be effectively loaded ontocommercially available heparin columns, e.g., the Hi-Trap heparin columnfrom Amersham Biosciences used by others (Zahn, A and Allain, J.-P.2005, J. Gen. Virol. 86, 677-685) for lab-scale purification ofHepatitis C and B viruses. The Vaccinia virion volume is approximately125 times larger than Hepatitis virion. (The diameter of the Vacciniavirus is, thus, appr. 250 nm as compared with the hepatitis C and Bvirions diameter being appr. 50 nm). Thus, available matrices as, e.g.,used in the column-based approach may not allow for adequate entrance ofvirions into the matrix, loading of sufficient amounts of virusparticles or sufficiently rapid flow through the column to meet theneeds for industrial scale purification. Zahn and Allain worked withvirus load up to 1×10⁶ in up to 1.0 ml volume. For pilot-scalepurification to achieve sufficient material for early clinical trialsvirus loading capacity higher than 1×10¹¹, preferably up 1×10¹³, involumes higher than 5 L, preferably up to 50 L, is needed. Forindustrial purification of Vaccinia virus loading capacity higher than1×10¹³, preferably higher than 1×10¹⁴ in volumes higher than 300 L,preferably higher than 600 L, is needed.

The large size of the Vaccinia virus may prevent effective steric accessbetween the specific surface proteins of the virions and the ligandimmobilized to the matrix. Currently described lab-scale methods of usefor purification of small virus particles may therefore not beindustrially applicable to purification of Vaccinia virus.

Due to the high number of functional surface molecules interacting withthe ligand used for binding of the Vaccinia virus particles, elution ofbound Vaccinia virus may require more harsh and therefore potentiallydenaturing conditions to elute and recover the Vaccinia virus particlesin a biologically effective form in high yields. The matrix, the liganddesign, the method of ligand immobilization, and the ligand density maytherefore require careful design to mediate an effective binding of theVaccinia virus and to permit an effective elution of biologically activeVaccinia virus particles.

Vaccinia virions are too large to be sterile filtered. The method usedin this invention has therefore been developed by to be applicable foran aseptic industrial-scale manufacturing process in a way ensuring fullcompliance with regulatory requirements regarding sterility of vaccines.In line with the above and for the purpose of this invention, the columnsubstituted with the ligand should be applicable forsterilization-in-place or should be available as a pre-sterilized unit.

The introduction of cell culture-derived smallpox vaccines required anadaptation of the original downstream processing schemes. Currentsmallpox vaccines like e.g. ACAM2000 are purified mainly after celldisruption by centrifugation and filtration methods (Abdalrhman et al.,Vaccine 24(19):4152-4160, 2006; Greenberg and Kennedy, Expert Opinion onInvestigational Drugs 17(4):555-564, 2008; and Monath et al.,International Journal of Infectious Diseases 8(Supplement 2):31-44,2004). The disadvantage of these methods is the limited depletion ofcontaminants like host cell DNA and proteins. In order to comply withregulatory expectations for current human smallpox vaccines based oncontinuous cell lines a nuclease treatment for host cell DNA depletionis often included in these processes (Greenberg and Kennedy, 2008; andMonath et al. 2004).

Examples of glycosaminoglycans in affinity chromatography applicationsare heparin and heparan sulfate. These are highly charged, linear andsulfated polysaccharides composed of repeating disaccharide unitscontaining an uronic acid (glucuronic or iduronic acid) and anN-sulfated or N-acetylated glucosamine (Ampofo et al., AnalyticalBiochemistry 199(2):249-255, 1991; Nugent, Proceedings of the NationalAcademy of Sciences of the United States of America 97(19):10301-10303,2000; Rabenstein, Nat. Prod. Rep. 19:312-331, 2002).

Cellufine® sulfate and sulfated cellulose membranes are sulfated glucosepolymers. Several studies reported antiviral activities of sulfatedcellulose and sulfated dextran/dextrines (Baba et al., Antimicrob.Agents Chemother. 32(11):1742-1745, 1988; Chattopadhyay et al.,International Journal of Biological Macromolecules 43(4):346-351, 2008;Mitsuya et al., Science 240(4852):646-649, 1988; Piret et al., J. Clin.Microbiol. 38(1):110-119, 2000), as well as the binding of virusparticles to Cellufine® sulfate (O'Neil et al., Bio/Technology11:173-178, 1993; Opitz et al., Biotechnol. and Bioeng.103(6):1144-1154, 2009). The precise interaction between these virusesand sulfated cellulose is currently not fully understood.

To achieve a bio-specific purification of Vaccinia virus particles withhigh biological activity, there is a need in the art for development ofindustrially usable ligands identical to or very similar to the presumednative ligand for Vaccinia target cell entry. Thus, use of a liganddisplaying highly specific and highly effective binding to the Vacciniavirus would be advantageous as it would improve purification by itsability to specifically sort out biologically active Vaccinia virusparticles thereby increasing the purity, viability, and functionality ofthe purified Vaccinia virus.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses methods for purifying viruses. The applicationof adsorption chromatography to capture cell-derived Vaccinia virusparticles after cell homogenization and cell debris clearance isdescribed. The invention includes virus purification using ion exchangeand pseudo-affinity chromatography, preferably based on heparin andsulfated cellulose.

The ability of different ion exchange and pseudo-affinity membraneadsorbers to capture cell-derived Vaccinia virus after cellhomogenization and clarification has been evaluated. In parallel, theoverall performance of classical bead-based resin chromatography(Cellufine® sulfate and Toyopearl® AF-Heparin) was investigated. The twotested pseudo-affinity membrane adsorbers (i.e. sulfated cellulose andheparin) were superior over the applied ion exchange membrane adsorberin terms of virus yield and contaminant depletion. Furthermore, studiesshowed an increase in productivity resulting from the increased volumethroughput of membrane adsorbers compared to classical bead-based columnchromatography methods. Overall virus recovery was approximately 60% forboth pseudo-affinity membrane adsorbers and the Cellufine® sulfateresin. Depletion of total protein ranged between 86% and 102% for alltested matrices. Remaining dsDNA in the product fraction varied between24% and 7% for the pseudo-affinity chromatography materials. Cellufine®sulfate and the reinforced sulfated cellulose membrane adsorbersachieved the lowest dsDNA product contamination. Finally, by acombination of pseudo-affinity with anion exchange membrane adsorbers afurther reduction of host cell DNA was achieved.

The invention encompasses methods for purifying biologically activeVaccinia viruses. In one embodiment, the method comprises loading asolid-phase matrix, to which a ligand is attached, with a biologicallyactive Vaccinia virus contained in a liquid-phase culture, washing thematrix; and eluting the biologically active Vaccinia virus. Preferably,the matrix with attached ligand is a sulfated cellulose. In oneembodiment, the solid-phase matrix comprises or is a membrane. In apreferred embodiment, the solid-phase matrix comprises or is a sulfatedreinforced cellulose membrane.

In one embodiment, the method is an industrial-scale process. In oneembodiment, the method is aseptic.

In a preferred embodiment, the eluted Vaccinia virus contains less than10 ng host-cell DNA per 10⁸ virus particles.

In a preferred embodiment, the Vaccinia virus is a recombinant Vacciniavirus. In a particularly preferred embodiment, the Vaccinia virus is MVAor recombinant MVA.

In one embodiment, the matrix comprises a pore size of greater than 0.25μm.

In one embodiment, contaminants are removed from the Vaccinia virus inthe liquid-phase culture.

In one embodiment, the Vaccinia virus is eluted with sodium chloride(NaCl). In a preferred embodiment, the Vaccinia virus is eluted by anincreasing NaCl concentration gradient ranging from 0.15 M to 2.0 M.Preferably, the Vaccinia virus is eluted with 2.0 M NaCl.

In one embodiment, a purification step by ion-exchange is included. In apreferred embodiment, the purification step by ion-exchange comprises amembrane.

In a preferred embodiment, the method reduces the amount of dsDNA in theeluted virus to less than 5% of input. In a particularly preferredembodiment, the method reduces the amount of dsDNA in the eluted virusto less than 0.1% of input.

In a preferred embodiment, the pH value of the virus preparation isadjusted to a pH ranging from 4.0-11.0.

In one embodiment, the eluted Vaccinia virus is administered to ananimal, preferably a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C depict the relative amounts of virus (A; ELISA), total dsDNA(B; PicoGreen®) and total protein (C; Pierce® BCA protein assay) duringthe capturing of CEF cell-derived MVA-BN® using anion exchange MA Q75-MAand D75-MA (15 layers, d=25 mm, A=75 cm2), 3 serially connected cationexchangers C75-MA (3×15 layers, d=25 mm, A=225 cm2), 3 seriallyconnected heparin-MA (3×15 layers, d=25 mm, A=225 cm2), SC-MA (15layers, d=25 mm, A=75 cm2) and bead-based resin Cellufine® sulfate (3 mlfixed bed, Tricorn 5/150 column). Adsorption buffer: 100 mM citric acid,pH 7.2; elution buffer: 100 mM citric acid, 2 M NaCl, pH 7.2). Thenumber of experiments is indicated in brackets in FIG. 1A; error bars:mean and standard deviation of each test series.

FIG. 2 depict the purification of MVA-BN® by a combination of a SC-MA(15 layers, d=25 mm, A=75 cm2) or heparin-MA (3×15 layers, d=25 mm,A=225 cm2) with an anion exchange MA Q75-MA (15 layers, d=25 mm, A=75cm2). The equilibration buffer was for all cases 100 mM citric acid pH7.2 and the elution buffer 100 mM citric acid+2 M NaCl pH 7.2. The flowrates for the adsorption process for the pseudo-affinity MA was 10ml/min and the flow rate for desorption 0.5 ml/min. The adsorption anddesorption flow rate of the Q75-MA was 0.5 ml/min. The relative viruscontent (green) was monitored by an ELISA; relative amounts of totalprotein (blue) and dsDNA (red) were quantified by the Pierce® BCAprotein assay and the Quant iT™ PicoGreen® assay, respectively. ELISAand total protein analysis of individual samples were conducted intriplicates and the dsDNA measurements in duplicates; (n) indicates thenumber of chromatographic experiments.

DETAILED DESCRIPTION OF THE INVENTION

In particular, the present invention is directed to a method for thepurification of biologically active Vaccinia virus comprising:

a. loading a solid-phase matrix, to which a ligand is attached, with aVaccinia virus contained in a liquid-phase culture;

b. washing the matrix, and

c. eluting the virus.

The ligand is a substance that, on the one hand, can be attached to thesolid-phase matrix, e.g., by binding or coupling thereto and that, onthe other hand, is able to form a reversible complex with the Vacciniavirus. Thus, by interacting with the virus, the virus is reversiblyretained. The ligand can be a biological molecule as, for example, apeptide and/or a lectin and/or an antibody and/or, preferably, acarbohydrate. The ligand may also comprise or consist of sulfate. In afurther embodiment, the ligand comprises one or more negatively chargedsulfate groups. Furthermore, the ligand can also be a hydrophobicmolecule as, for example, an aromatic phenyl group. The ligand can beattached to the matrix directly, e.g, by direct binding, or can beattached to the matrix indirectly though another molecule, e.g. bycoupling through a linker or spacer.

The solid-phase matrix can be a gel, bead, well, membrane, column, etc.In a preferred embodiment of the invention, the solid-phase comprises oris a membrane, in particular a cellulose membrane. However, a broadrange of other polymers modified with specific groups capable to bindthe virus can be used. Preferred are hydrophilic polymers. Examples arecellulose derivatives (cellulose esters and mixtures thereof, cellulosehydrate, cellulose acetate, cellulose nitrate); agarose and itsderivatives; other polysaccachrides like chitin and chitosan;polyolefines (polypropylene); polysulfone; plolyethersulfone;polystyrene; aromatic and aliphatic polyamides; polysulfonamides;halogenated polymers (polyvinylchloride, polyvinylfluoride,polyvinylidenfluoride); polyesters; homo- and copolymers ofacryInitrile.

The method and further embodiments of the invention can overcome thelimitations of currently known methods preventing industrial-scale,effective purification of Vaccinia virus particles with high biologicalactivity and purity. The method is superior in terms of yield, processtime, purity, recovery of biologically active Vaccinia virus particlesand costs to existing pilot-scale methods for purification of Vacciniavirus particles, which are primarily based on sucrose-cushioncentrifugation and/or diafiltration or non-specific ion-exchangechromatography. It is also superior in terms of yield, process time,purity, recovery of biologically active Vaccinia virus particles, andcosts to the only existing large-scale method for purification ofVaccinia virus particles, which is based on ultrafiltration, enzymaticDNA degradation, and diafiltration.

According to the present invention, Vaccinia virus can be purified underaseptic conditions to obtain a biologically active, stable, and highlypure virus preparation in high yield. The Vaccinia viruses can be nativeor recombinant.

The present invention provides an improved method for asepticpurification of Vaccinia viruses in lab-, pilot-, and, preferably, inindustrial-scale, leading to a biologically active, stable and highlypure virus preparation in high yield.

This invention provides a more time-effective and cost-effective processfor purification of Vaccinia viruses and recombinant Vaccinia viruses,Modified Vaccinia virus Ankara (MVA) and recombinant MVA, MVA-BN® andrecombinant MVA-BN®, leading to a biologically active, stable and highlypure virus preparation in high yield.

In another embodiment, this invention provides virus preparationsproduced by the method of the invention.

Use of the eluted Vaccinia virus or recombinant Vaccinia virus, orModified Vaccinia virus Ankara (MVA) or recombinant MVA or MVA-BN® orrecombinant MVA-BN®, all preferably obtained by the method according tothe present invention, for the preparation of a pharmaceuticalcomposition, in particular a vaccine, is also an embodiment of theinvention. The virus and/or pharmaceutical preparation is preferablyused for the treatment and/or the prevention of cancer and/or of aninfectious disease.

A method for inducing an immune response or for the vaccination of ananimal, specifically of a mammal, including a human, in need thereof,characterized by the administration of a Vaccinia virus or recombinantVaccinia virus, or Modified Vaccinia virus Ankara (MVA) or recombinantMVA or MVA-BN® or recombinant MVA-BN® vaccine prepared by a processcomprising a purification step as described above is a furtherembodiment of the invention.

As used herein, an “attenuated virus” is a strain of a virus whosepathogenicity has been reduced compared to its precursor, for example byserial passaging and/or by plaque purification on certain cell lines, orby other means, so that it has become less virulent because it does notreplicate, or exhibits very little replication, but is still capable ofinitiating and stimulating a strong immune response equal to that of thenatural virus or stronger, without producing the specific disease.

According to a further preferred embodiment of the present invention,glucosamine glycan (GAG), in particular heparan sulfate or heparin, or aGAG-like substance is used as ligand.

As used herein, “glycosaminoglycans” (GAGs) are long un-branchedpolysaccharides consisting of a repeating disaccharide unit. Some GAGsare located on the cell surface where they regulate a variety ofbiological activities such as developmental processes, bloodcoagulation, tumor metastasis, and virus infection.

As used herein, “GAG-like agents” are defined as any molecule which issimilar to the known GAGs, but can be modified, for example, by theaddition of extra sulfate groups (e.g. over-sulfated heparin). “GAG-likeligands” can be synthetic or naturally occurring substances.Additionally, the term “GAG-like ligands” also covers substancesmimicking the properties of GAGs as ligands in ligand-solid-phasecomplexes. One example for a “GAG-like ligand” mimicking GAG,specifically heparin, as ligand is Sulfate attached to ReinforcedCellulose as solid-phase, thus forming Sulfated Reinforced Cellulose(SRC) as ligand-solid-phase complex. The use of SRC complex is also apreferred embodiment of the present invention. Stabilized ReinforcedCellulose membranes can be obtained, for example, from Sartorius AG.

As used herein, “Bulk Drug Substance” refers to the purified viruspreparation just prior to the step of formulation, fill and finish intothe final vaccine.

As used herein, “Biological activity” is defined as Vaccinia virusvirions that are either 1) infectious in at least one cell type, e.g.CEFs, 2) immunogenic in humans, or 3) both infectious and immunogenic. A“biologically active” Vaccinia virus is one that is either infectious inat least one cell type, e.g. CEFs, or immunogenic in humans, or both. Ina preferred embodiment, the Vaccinia virus is infectious in CEFs and isimmunogenic in humans.

As used herein, “contaminants” cover any unwanted substances which mayoriginate from the host cells used for virus growth (e.g. host cell DNAor protein) or from any additives used during the manufacturing processincluding upstream (e.g. gentamicin) and downstream (e.g. Benzonase).

As used herein, “continuous cell culture (or immortalized cell culture)”describes cells that have been propagated in culture since theestablishment of a primary culture, and they are able to grow andsurvive beyond the natural limit of senescence. Such surviving cells areconsidered as immortal. The term immortalized cells were first appliedfor cancer cells which were able to avoid apoptosis by expressing atelomere-lengthening enzyme. Continuous or immortalized cell lines canbe created, e.g., by induction of oncogenes or by loss of tumorsuppressor genes.

As used herein, “heparan sulfate” is a member of the glycosaminoglycanfamily of carbohydrates. Heparan sulfate is very closely related instructure to heparin, and they both consist of repeating disaccharideunits which are variably sulfated. The most common disaccharide unit inheparan sulfate consists of a glucuronic linked to N-acetyl glucosamine,which typically makes up approx. 50% of the total disaccharide units.

As used herein, “heparin” is a member of the glycosaminoglycan family ofcarbohydrates. Heparin is very closely related in structure to heparansulfate, and they both consist of repeating disaccharide units which arevariably sulfated. In heparin, the most common disaccharide unitconsists of a sulfated iduronic acid linked to a sulfatedglucopyranosyl. To differentiate heparin from heparan sulfate, it hasbeen suggested that in order to qualify a GAG as heparin, the content ofN-sulfate groups should largely exceed that of N-acetyl groups and theconcentration of O-sulfate groups should exceed those of N-sulfate(Gallagher et al. 1985, Biochem. J. 230: 665-674).

As used herein, “industrial scale” or large-scale for the manufacturingof Vaccinia virus or recombinant Vaccinia virus-based vaccines comprisesmethods capable of providing a minimum of 50,000 doses of 1.0×10⁸ virusparticles (total minimum 5.0×10¹² virus particles) per batch (productionrun). Preferably, more than 100,000 doses of 1.0×10⁸ virus particles(total minimum 1.0×10¹³ virus particles) per batch (production run) areprovided.

As used herein, “lab-scale” comprises virus preparation methods ofproviding less than 5,000 doses of 1.0×10⁸ virus particles (total lessthan 5.0×10¹¹ virus particles) per batch (production run).

As used herein, “pilot-scale” comprises virus preparation methods ofproviding more than 5,000 doses of 1.0×10⁸ virus particles (total morethan 5.0×10¹¹ virus particles), but less than 50,000 doses of 1.0×10⁸virus particles (total minimum 5.0×10¹² virus particles) per batch(production run).

As used herein, “Primary cell culture”, refers to the stage where thecells have been isolated from the relevant tissue (e.g. from specificpathogen free (SPF) hens eggs), but before the first sub-culture. Thismeans that the cells have not been grown or divided any further from theoriginal origin.

As used herein, “Purity” of the Vaccinia virus preparation or vaccine isinvestigated in relation to the content of the impurities DNA, protein,Benzonase, and gentamicin. The purity is expressed as specific impurity,which is the amount of each impurity per dose (e.g. ng DNA/dose).

As used herein, “purification” of a Vaccinia virus preparation refers tothe removal or measurable reduction in the level of some contaminant ina Vaccinia virus preparation.

As used herein, “Recombinant Vaccinia virus” is a virus, where a pieceof foreign genetic material (from e.g. HIV virus) has been inserted intothe viral genome. Thereby, both the Vaccinia virus genes and anyinserted genes will be expressed during infection of the Vaccinia virusin the host cell.

As used herein, “Stability” means a measure of how the quality of thevirus preparation (Bulk Drug Substance (BDS) or Final Drug Product(FDP)) varies with time under the influence of a variety ofenvironmental factors such as temperature, humidity and lights, andestablishes a retest period for the BDS or a shelf-life for the FDP atrecommended storage conditions (Guidance for industry Q1A (R2).

As used herein, a “Virus preparation” is a suspension containing virus.The suspension could be from any of the following steps in amanufacturing process: after virus growth, after virus harvest, aftervirus purification (typically the BDS), after formulation, or the finalvaccine (FDP).

As used herein “vaccinia virus forms” refer to the three different typesof virions produced by infected target cells: Mature virions (MV),wrapped virions (WV), and extra-cellular virions (EV) (Moss, B. 2006,Virology, 344:48-54). The EV form comprises the two forms previouslyknown as cell-associated enveloped virus (CEV), and extra-cellularenveloped virus (EEV) (Smith, G. L. 2002, J. Gen. Virol. 83: 2915-2931).

The MV and EV forms are morphologically different since the EV formcontains an additional lipoprotein envelope. Furthermore, these twoforms contain different surface proteins (see Table 1), which areinvolved in the infection of the target cells by interaction withsurface molecules on the target cell, such as glycosaminglycans (GAGs)(Carter, G. C. et al. 2005, J. Gen. Virol. 86: 12791290). The inventioninvolves use of the purification of all forms of Vaccinia Virus.

The different forms of Vaccinia virions contain different surfaceproteins, which are involved in the infection of the target cells byinteraction with surface molecules on the target cell, such asglycosaminglycans (GAGs) (Carter, G. C. et al. 2005, J. Gen. Virol. 86:1279-1290). These surface proteins will as mentioned supra be referredto as receptors. On the MV form, a surface protein named p14 or A27L(the latter term will be used in this application) is involved in theinitial attachment of the virions to the target cell. A27L binds to GAGstructures on the target cell prior to entry into the cell (Chung C. etal. 1998, J. Virol. 72: 1577-1585), (Hsiao J. C. et al. 1998 J. Virol.72: 8374-8379), (Vazquez M. et al. 1999, J. Virol. 73: 9098-9109)(Carter G. C. et al. 2005, J. Gen. Virol. 86: 1279-1290). The naturalligand for A27L is presumed to be the GAG known as heparan sulfate (HS).Heparan Sulfate belongs to a group of molecules known asglycosaminglycans (GAGs). GAGs are found ubiquitously on cell surfaces.(Taylor and Drickamer 2006, Introduction to Glycobiology, 2^(nd)edition, Oxford University Press). GAGs are negatively charged moleculescontaining sulfate groups. The A27L protein is located on the surface ofthe virions and is anchored to the membrane by interaction with the A17Lprotein (Rodriguez D. et al. 1993, J. Virol. 67: 3435-3440) (Vazquez M.et al. 1998, J. Virol. 72: 10126-10137). Therefore, the interactionbetween A27L and Al17L can be kept intact during isolation in order toretain full biological activity of the virions. The specific nature ofthe protein-protein interaction between A17L and A27L has not been fullyelucidated, but it has been suggested that a presumed “Leucine-zipper”region in the A27L is involved in the interaction with A17L (Vazquez M.et al. 19981, J. Virol. 72: 10126-10137).

The invention encompasses the use of the affinity interaction betweenthe A27L surface protein on the MV form and glucosaminglycans, inparticular Heparan Sulfate, for purification of the MV form of VacciniaVirus.

The term “ligand”, thus, refers both to a receptor on a target cell andto the specific binding structure attached to a solid-phase matrix usedfor purification of Vaccinia.

The same principle as described above can be applied to interactionsbetween other target cell surface structures and other Vaccinia surfaceproteins of the MV form participating in the Vaccinia virus' recognitionof, attachment to, entry into and/or fusion with the target cell (seeTable 1). Other WV and EV surface proteins are summarized in Table 1.The entire A27L protein, or fragments thereof containing the bindingregion for the GAG ligand can be used as agents to elute Vacciniaviruses-GAG complexes from a solid-phase column of the invention.Fragments can be readily generated by routine molecular techniques andscreened for their ability to dissociate Vaccinia viruses-GAG complexesusing routine techniques known in the art, such as by measuring eluted,biologically active virus.

The presumed native GAG-ligand for the MV form of Vaccinia is HeparanSulfate (HS) and can be one of the suitable ligands. The invention alsocomprises use of “non-native” ligands for purification of Vacciniavirus. Such non-native ligands are compounds with a high degree ofstructural and/or conformational similarity to native ligands. As anexample, Heparin, which is a close analogue to the native ligand forA27L, HS, can be used for affinity-purification of MV form byinteraction with the A27L surface protein, see further below. Heparinhas been shown to partially inhibit the binding between target cells andVaccinia virus and can therefore also be used for affinity purificationof the MV form of Vaccinia. Other GAG-ligands and GAG-like ligands canalso be used.

In one embodiment of the invention, Heparan Sulfate, used for affinitypurification of the MV form of Vaccinia, binds A27L on biologicallyactive Vaccinia viruses, but does not bind inactive Vaccinia viruses orVaccinia virus fragments.

The ligand makes possible the elution of the bound Vaccinia virus undersuch mild conditions that the Vaccinia virus fully retain theirbiologically activity. This means that the structure of A27L and theinteraction between A27L and A17L can be kept intact.

The binding and elution characteristics for the GAG-ligand substitutedmatrix depend not only on the individual characteristics of the matrixand ligand, but also on the interplay between the two.

By modifying e.g. the ligand density or by attaching, e.g. binding orcoupling of, the ligand to the matrix by “arms” or “spacers” ofdifferent length and chemical characteristics (hydrophobicity,hydrophilicity) the binding strength between the target GAG-ligandstructure and the A27L surface protein on the Vaccinia virus can bealtered, which can be used to e.g. enhance the capture or ease theelution.

To enhance the purification method, the matrix in the form of achromatography gel or membrane to be used for the purificationpreferably:

-   -   Has a high pore size (to make as many ligands as possible        accessible to the Vaccinia virus)    -   Has a rigid structure to allow for fast flow rates    -   Is available in a form permitting direct or indirect attachment,        e.g. by binding or coupling, of ligands    -   Is applicable for sterilization in place or available as a        pre-sterilized unit, e.g. by using radiation.

In one embodiment, the solid phase matrix is a gel or membrane with apore size of 0.25 μm, preferably of more than 0.25 μm, more preferablyof 1.0-3.0 μm demonstrating a linear flow rate under actual purificationconditions of 10 cm/min, preferably 20 cm/min. The pore size of thematrix can be 0.25-0.5 μm, 0.5-0.75 μm, 0.75-1.0 μm, 1.0-2.0 μm, 2.0-3.0μm, or greater than 3.0 μm.

In one embodiment, with the solid phase matrix containing a heparansulfate as an immobilized ligand, the virus harvest from the upstreamvirus growth process is loaded in a crude (unpurified) form with a flowrate of 10 cm/min, preferably 20 cm/min at a virus concentration of 10⁶virions per mL in pilot scale and 10⁷ virions per mL in industrialscale.

In one embodiment, there are three steps in the purification process ofthe invention, which are common for most affinity chromatographyprocesses:

-   -   1) Loading of Vaccinia virus or Vaccinia recombinant virus onto        the solid phase;    -   2) Washing of the solid phase to remove contaminants; and    -   3) Elution of the Vaccinia virus or recombinant virus to be        isolated.        Step 1. Loading of Vaccinia Virus or Recombinant Virus onto a        Solid-phase Matrix

Loading to the solid phase with, e.g., Heparane Sulphate or another GAGor GAG-like structure attached as ligand, can be performed by a batch-,column- or membrane approach.

The membrane approach can have some benefits, specifically for largebio-molecules, in particular for large viruses like Vaccinia viruses:For example, large pore sizes and the availability of the ligand on thesurface of the membrane allow high binding capacities of even largeviral particles. The membrane approach is, thus, a preferred embodimentof the present invention.

In all embodiments mentioned above, the Vaccinia virus or recombinantvirus to be isolated is present in a liquid phase. When the Vacciniavirus or recombinant virus gets close to the GAG or GAG-like ligand theVaccinia virus will bind specifically to or be “captured by” theGAG-ligand, thereby the Vaccinia virus or recombinant Vaccinia virus canbe temporarily immobilized on the solid phase, while the contaminantswill remain in the liquid phase.

By appropriate selection of the ligand type, ligand density and ligandsteric configuration, the binding parameters of Vaccinia virus via A27Lsurface protein to the column can be altered, thereby providing meansfor optimization of the purification parameters.

Step 2. Washing of the Solid Phase to Remove Contaminants

When the binding of the biologically active Vaccinia viruses orrecombinant viruses to the ligand has proceeded sufficiently, the hostcell contaminants (in particular host cell DNA and proteins) that remainin the liquid phase can be removed by washing the solid phase, to whichthe Vaccinia virus is bound, with an appropriate washing medium.

Step 3. Eluting the Vaccinia Virus or Recombinant Virus by Specific orNon-specific Agents

The biologically active Vaccinia viruses or recombinant viruses can beeluted. The elution of the captured Vaccinia virus can be performed, forexample, by:

Agents specifically disrupting the specific interaction between, e.g.,the GAG-ligand and the A27L surface protein on the Vaccinia virus (to becalled specific agents), or by

Agents non-specifically disrupting the electrostatic interactionbetween, e.g., the negatively charged GAG-ligand and the positivelycharged A27L surface protein (to be called non-specific agents).

According to further embodiments of the present invention, the Vacciniavirus is eluted with GAG or a GAG-like ligand or part thereof, with theGAG-binding domain of A27L or part thereof, and/or with anO-glycoside-binding cleaving enzyme.

Elution of the virus is, further, preferably performed with sodiumchloride, more preferably by an increasing NaCl concentration gradientranging from 0.15 M to 2.0 M.

Pre-treatment

Prior to loading on the solid phase, a pre-treatment of the virussuspension can be performed, specifically in order to removecontaminants from the Vaccinia virus in the liquid-phase culture.

Pre-treatment can be one or more of the following steps either alone orin combination:

-   -   1) Homogenization of the host cells    -   Ultrasound treatment    -   Freeze/thaw    -   Hypo-osmotic lysis    -   High-pressure treatment    -   2) Removal of cell debris    -   Centrifugation    -   Filtration    -   3) Removal/reduction of host cell DNA    -   Benzonase treatment    -   Cationic exchange    -   Selective precipitation by cationic detergents

According to a further embodiment of the invention, the pH value of theviral suspension is decreased just prior to loading in order to improvethe binding of the virus particle to the ligand. The pH value of theviral suspension can be decreased from appr. pH 7.0-8.0 to 4.0-6.9, inparticular to pH 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.5, 5.6,5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 6.9. Preferably, the pH value isdecreased from pH 7.0-8.0 to pH 5.8. Subsequently, just after loadingand before elution, the pH value is again increased to pH 7.0-8.0, inparticular to pH 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, preferably to pH7.7, in order to improve the stability of the viral particles.

Post-treatment

Depending on the agent used for elution of the Vaccinia virus orrecombinant virus, post-treatment can be performed to enhance the purityof the virus preparation. The post-treatment could beultra/diafiltration for further removal of impurities and/or specific ornon-specific agents used for elution. To obtain an efficientpurification of the virus, it is also preferred to combine thepurification according to the invention with one or more furtherpurification steps, e.g., by ion-exchange(s). Ion-exchange(s) can, then,also be performed as post-treatment step(s).

In order to prevent aggregation of the purified virus suspension and,thus, to, inter alia, improve the detection of infectious particles, inparticular by the TCID50 method, it can also be suitable to increase thepH value after elution of the virus, in particular to a pH value of upto 9 or more, in particular to pH 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5,8.6, 8.8, 9.0, 9.2, 9.4, 9.5, 9.6, 9.8, 10.0, 10.2, 10.4, 10.5.Preferably, the pH value is increased from, in particular, pH 7.0, 7.2,7.4, 7.5, 7.6, 7.8, 8.0, preferably pH 7.7, to pH 9.0.

According to a further embodiment of the present invention, the Vacciniavirus sample contains host-cell DNA in the range of 10-20 μg per dose(1×10⁸ TCID₅₀−3.2×10⁸ TCID₅₀), preferably 10 ng, more preferably lessthan 10 ng host-cell DNA per 10⁸ virus particles after performance ofthe purification steps according to the invention, i.e, after elution ofthe virus.

Preferably, the amount of host-cell DNA in a VV dose of 1×10⁸ TCID₅₀ is10-20 μg, 1-10 μg, 100 ng-1 μg, 10-100 ng, or 1-10 ng. The amount ofdsDNA in a VV sample can be reduced by the purification method to lessthan 40%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of input.

Preferably, amount of protein in the purified VV is less than 250 μg/ml,100 μg/ml, 50 μg/ml g, 20 μg/ml, 10 μg/ml, or 5 μg/ml.

The practice of the invention employs techniques in molecular biology,protein analysis, and microbiology, which are within the skilledpractitioner of the art. Such techniques are explained fully in, forexample, Ausubel et al. 1995, eds, Current Protocols in MolecularBiology, John Wiley & Sons, New York.

Modifications and variations of this invention will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by the way of example only, and the invention is not to beconstrued as limited thereby.

In one embodiment, the invention provides a more time-effective andcost-effective process for purification of Vaccinia viruses andrecombinant-modified Vaccinia viruses in higher yield, comprising one ormore of the following steps:

-   -   a. loading a solid-phase matrix with a liquid-phase virus        preparation, wherein the solid-phase matrix comprises a ligand        appropriate for interacting with the virus, e.g. by reversibly        binding the virus    -   b. washing of the matrix, and    -   c. eluting the virus.

Additional aspects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention.

In a preferred embodiment, the method comprises the following steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix comprising one or more appropriate virus-binding ligands        with a liquid-phase virus preparation,    -   b. Washing of the matrix with an appropriate solvent to remove        contaminants, and    -   c. Eluting the Vaccinia virus with an appropriate solvent to        achieve a highly pure, biologically active, stable virus        preparation.

In a further preferred embodiment, the method comprises the followingsteps:

a. Loading a column, membrane, filter or similar solid-phase matrixcomprising one or more appropriate glucosamine glycan (GAG) or GAG-likevirus-binding ligands with a liquid-phase virus preparation

-   -   b. Washing of the matrix with an appropriate solvent to remove        contaminants, and    -   c. Eluting the Vaccinia virus with a solvent resulting in an        concentration gradient of a non-specific eluent such as NaCl, H+        or of specific eluent such as a GAG-like compound or and A27L        peptide or peptide-fragment to achieve a highly pure,        biologically active, stable virus preparation.

In one particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparin (HP) with a Vaccinia virus        preparation dissolved in a neutral buffer (pH 6.5 to 8.5,        preferably >=pH 7.5) with a physiological salt concentration        (approximately 150 mM NaCl),    -   b. Washing of the matrix with a sufficient amount of the loading        buffer to ensure complete elution of all non-binding Vaccinia        virus particles and non-binding contaminants, and    -   c. Eluting the Vaccinia virus with an increasing concentration        of NaCl, from 0.15 to 2.0 M NaCl, to initially remove        contaminants with less affinity than the Vaccinia virus        particles and to finally elute the biologically active Vaccinia        virus particles.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparin (HP) with a Vaccinia virus        preparation dissolved in a neutral buffer (pH 6.5 to 8.5,        preferably >=pH 7.5) with a physiological salt concentration        (approximately 150 mM NaCl). An appropriate buffer is Phosphate        Buffered Saline (PBS), e.g. 0.01 to 0.1 M phosphate, 0.15 M        NaCl, pH 7.5. Other appropriate buffers are Tris-NaCl, e.g. 0.01        to 0.1 M Tris, 0.15 M    -   b. Washing of the matrix with a sufficient amount of the loading        buffer e.g. PBS (0.01 M phosphate, 0.15 M NaCl, pH 7.5) to        ensure complete elution of all non-binding Vaccinia virus        particles and nonbinding contaminants, as measured by the return        of the 280 nm absorbance signal to the pre-loading baseline, and    -   c. Eluting the Vaccinia virus with an increasing concentration        of NaCl in PBS, starting with 0.15 M and ending with 2.0 M NaCl.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparin (HP) with a Vaccinia virus        preparation dissolved in a neutral buffer (pH 6.5 to 8.5,        preferably >=pH 7.5) with a physiological salt concentration        (approximately 150 mM NaCl). An appropriate buffer is Phosphate        Buffered Saline (PBS), e.g. 0.01 to 0.1 M phosphate, 0.15 M        NaCl, pH 7.5. Other appropriate buffers are Tris-NaCl, e.g. 0.01        to 0.1 M Tris, 0.15 M NaCl, pH 8.0, and HEPES-NaCl, e.g. 0.01 to        0.1 M HEPES, 0.15 M NaCl, pH 7.5,    -   b. Washing (Wash 1) of the matrix with a sufficient amount of        the loading buffer e.g. PBS (0.01 M phosphate, 0.15 M NaCl, pH        7.5) to ensure complete elution of all non-binding Vaccinia        virus particles and non-binding contaminants, as measured by the        return of the 280 nm absorbance signal to the pre-loading        baseline,    -   c. Washing (Wash 2) of the matrix with an additional washing        buffer e.g. Glycine Buffered Saline (GBS) 0.02 M, 0.15 M NaCl,        pH 9.0) to remove loosely bound contaminants, and    -   d. Eluting the Vaccinia virus with an increasing concentration        of NaCl in GBS 0.02 M pH 9.0, starting with 0.15 M and ending        with 2.0 M NaCl.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparane Sulphate (HS) with a Vaccinia        virus preparation dissolved in a neutral buffer (pH 6.5 to 8.5,        preferably >=pH 7.5) with a physiological salt concentration        (approximately 150 mM NaCl). An appropriate buffer is Phosphate        Buffered Saline (PBS), e.g. 0.01 to 0.1 M phosphate, 0.15 M        NaCl, pH 7.5. Other appropriate buffers are Tris-NaCl, e.g. 0.01        to 0.1 M Tris, 0.15 M    -   b. Washing of the matrix with a sufficient amount of the loading        buffer e.g. to ensure complete elution of all non-binding        Vaccinia virus particles and non-binding contaminants, as        measured by the return of the 280 nm absorbance signal to the        pre-loading baseline, and    -   c. Eluting the Vaccinia virus with an increasing concentration        of Low Molecular Weight Heparin, 0.01 to 0.5 M, in PBS 0.1 M,        NaCl 0.15 M, pH 7.5.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparane Sulphate (HS) with a Vaccinia        virus preparation dissolved in Phosphate Buffered Saline (PBS),        0.02 M phosphate, 0.15 M NaCl, pH 7.5,    -   b. Washing of the matrix with a sufficient amount of the loading        buffer e.g. to ensure complete elution of all non-binding        Vaccinia virus particles and non-binding contaminants, as        measured by the return of the 280 nm absorbance signal to the        pre-loading baseline, and    -   c. Eluting the Vaccinia virus with an increasing concentration        of an HS-derived oligosaccharide. The basic repeating        disaccharide unit in HS-derived oligosaccharide is a,(31-        4-linked sequence of glucosamine and uronic acid. The        glucosamine residues are either N-acetylated (GlcNAc) or        N-sulphated (GlcNSO3-). Other monosaccharide residues e.g.        iduronic acid and substitutions may occur, e.g. 2-0-sulphated        iduronic acid. The oligosaccharide consists of 2 to 10 repeating        disaccharide units. The oligosaccharide concentration used for        elution of the Vaccinia virus particles runs from 0.01 M to 0.5        M in PBS, 0.02 M phosphate, 0.15 M NaCl, pH 7.5.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparane Sulphate (HS) with a Vaccinia        virus preparation dissolved in Phosphate Buffered Saline (PBS),        0.02 M phosphate, 0.15 M NaCl, pH 7.5,    -   b. Washing of the matrix with a sufficient amount of the loading        buffer e.g. to ensure complete elution of all non-binding        Vaccinia virus particles and non-binding contaminants, as        measured by the return of the 280 nm absorbance signal to the        pre-loading baseline, and    -   c. Eluting the Vaccinia virus with an increasing concentration        of an Vaccinia virus particles surface protein or a peptide or        peptide-fragment derived hereof. The preferred surface protein        is A27L, the preferred peptide is A27L, and the preferred A27L        peptide-fragment is fragment containing 4-10 amino acid residues        of the A27L peptide sequence responsible for the binding between        A27L and the HS. The peptide concentration used for elution of        the Vaccinia virus particles runs from 0.01 M to 0.5 M in PBS,        0.02 M phosphate, 0.15 M NaCl, pH 7.5.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

-   -   a. Loading a column, membrane, filter or similar solid-phase        matrix substituted with a Heparane Sulphate (HS) with a Vaccinia        virus preparation dissolved in Phosphate Buffered Saline (PBS),        0.02 M phosphate, 0.15 M NaCl, pH 7.5,    -   b. Washing of the matrix with a sufficient amount of the loading        buffer e.g. to ensure complete elution of all non-binding        Vaccinia virus particles and non-binding contaminants, as        measured by the return of the 280 nm absorbance signal to the        pre-loading baseline, and    -   c. Eluting the Vaccinia virus with an enzyme capable of        partially cleaving one or more glycoside linkages between the        repeating disaccharide units, inside the repeating disaccharide        unit or elsewhere in the HS molecule. Preferred enzymes are        Heparin Lyase I, II and III. The elution is performed by        saturation of the column with the enzyme solution. After an        appropriate digestion time the unbound complex of Vaccinia virus        particles and GAG-residues bound to the Vaccinia virus particles        is eluted with PBS, 0.02 M phosphate, 0.15 M NaCl, pH 7.5. The        Vaccinia virus particle-GAG-residue complex is dissociated with        a mild NaCl solution e.g. PBS 0.02 M, 0.15 M NaCl, pH 7.5 and        the GAG-residues are removed by diafiltration.

EXAMPLES

Affinity purifications are made applying either column chromatographywith e.g. Toyopearls or membrane chromatography using e.g. a membrane(e.g. Sartobind MA 75 (Sartorius)) both of which are substituted with aGAG-ligand (e.g. Heparin or Heparan Sulfate) or a GAG-like ligand.

The below mentioned examples are all in lab-scale.

Example 1

Two ml of a highly concentrated and previously purified Vaccinia viruspreparation with approximately 2×10⁹ virus particles per ml were appliedto a column with packed with Toyopearl AF-Heparin.

The column was washed with PBS 0.01 M, 0.15 M NaCl, pH 7.2. The A280absorbance signal used for monitoring of Vaccinia virus particle andhost cell protein concentrations returned to baseline (the pre-loadingvalue) after 12 minutes. The washing continued for a total of 25minutes.

The bound Vaccinia virus particles were eluted by a NaCl concentrationgradient in PBS 0.01 M, pH 7.2. The concentration of NaCl was increasedlinearly from 0.15 M to 2.0 M. The elution started after approximately atotal of 30 minutes (5 minutes after starting the gradient). The majorpeak was eluted 7 minutes later (at T=37 minutes). The peak contained ahigh concentration of Vaccinia virus particles as assessed by the LaserScattering signal used for monitoring of Vaccinia virus particles. Theelution was completed after approximately 25 minutes (T=55 minutes).

The eluate was analyzed by a Vaccinia Virus specific ELISA showing avirus recovery rate of appr. 70%-90%. Host cell protein was analysed byuse of the BCA total protein assay showing appr. 10% of protein in theeluate. Host cell DNA was analysed by a total DNA assay showing anadditional removal of DNA of appr. 40% in the wash and flow-through.

Example 2

Two ml of a highly concentrated and previously purified Vaccinia viruspreparation with approximately 2×10⁹ virus particles per ml was appliedto a Sartobind MA75 Heparin membrane.

The membrane was washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal used for monitoring of Vaccinia virus particle andhost cell protein concentrations returned to baseline (the pre-loadingvalue) after 12 minutes. The washing continued for a total of 16minutes.

The bound Vaccinia virus particles were eluted by a NaCl concentrationgradient in PBS 0.01 M, pH 7.5. The concentration of NaCl was increasedlinearly from 0.15 M to 2.0 M. The elution started after approximately atotal of 20 minutes (4 minutes after starting the gradient). The majorpeak was eluted 5 minutes later (at T=25 minutes). The peak contained ahigh concentration of Vaccinia virus particles as assessed by the LaserScattering signal used for monitoring of Vaccinia virus particles.

The eluate was analyzed by a Vaccinia Virus specific ELISA showing avirus recovery rate of appr. 55%. Host cell protein was analyzed by useof the BCA total protein assay and revealed a protein recovery of appr.5% in the eluate. Host cell DNA was analyzed by a total DNA assay andrevealed appr. 10% DNA in the eluate.

Example 3

Two ml of a highly concentrated Vaccinia virus preparation withapproximately 2×10⁹ virus particles per ml are applied to a SartobindMA75 Heparin membrane.

The membrane is washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal is used for monitoring of Vaccinia virus particle andthe host cell protein concentrations until it returns to baseline (thepre-loading value). The washings are continued for a total of 20minutes.

The bound Vaccinia virus particles are eluted by a pH concentrationgradient in GBS 0.02 M, 0.15 M NaCl. The initial pH is 8.5, increasingto pH 10.5. The concentration of NaCl is increased linearly from 0.15 Mto 2.0 M.

The eluate is analyzed by titration for viable (infectious) Vacciniavirus particles by a Tissue Culture cytopatic effect assay (TCID50), fortotal number of Vaccinia virus particles by a real-time qPCR forVaccinia DNA, for host cell protein by use of the BCA total proteinassay and for host cell DNA by use of a real-time qPCR. The recovery canbe >60% and biological activity of the recovered Vaccinia virus can be>75%.

Example 4

Two ml of a highly concentrated Vaccinia virus preparation withapproximately 2×10⁹ virus particles per ml are applied to a SartobindMA75 Heparin membrane.

The membrane is washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal is used for monitoring of Vaccinia virus particle andthe host cell protein concentrations until it returns to baseline (thepre-loading value). The washings are continued for a total of 20minutes.

The elution is performed with a concentration gradient of low-molecularweight heparin (LMW-HP) in PBS 0.1 M, 0.15 M NaCl, pH 7.5. The gradientis run from 0.01 to 0.5 M LMW-Heparin.

The eluate is analyzed by titration for viable (infectious) Vacciniavirus particles by a Tissue Culture cytopathic effect assay (TCID50),for total number of Vaccinia virus particles by a real-time qPCR forVaccinia DNA, for host cell protein by use of the BCA total proteinassay and for host cell DNA by use of a real-time qPCR. The recovery canbe >70% and biological activity of the recovered Vaccinia virus can be>80%.

Example 5

Two ml of a highly concentrated Vaccinia virus preparation withapproximately 2×10⁹ virus particles per ml are applied to a SartobindMA75 Heparin membrane.

The membrane is washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal is used for monitoring of Vaccinia virus particle andthe host cell protein concentrations until it returns to baseline (thepreloading value). The washings are continued for a total of 20 minutes.

The elution is performed with a concentration of a gradient of A27Lpeptide (A27LP) in PBS 0.1 M, 0.15 M NaCl, pH 7.5. The gradient is runfrom 0.01 to 0.5 M A27LP.

The eluate is analyzed by titration for viable (infectious) Vacciniavirus particles by a Tissue Culture cytopatic effect assay (TCID50), fortotal number of Vaccinia virus particles by a real-time qPCR forVaccinia DNA, for host cell protein by use of the BCA total proteinassay and for host cell DNA by use of a real-time qPCR. The recovery canbe >70% and biological activity of the recovered Vaccinia virus can be>80%.

Example 6

Two ml of a highly concentrated Vaccinia virus preparation withapproximately 2×10⁹ virus particles per ml are applied to a SartobindMA75 Heparin membrane.

The membrane is washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal is used for monitoring of Vaccinia virus particle andthe host cell protein concentrations until it returns to baseline (thepre-loading value). The washings are continued for a total of 20minutes.

The elution is performed with a glycoside linkage cleaving enzymeHeparin Lyase in PBS 0.1 M, 0.15 M NaCl, pH 7.5. The membrane issaturated with Heparin Lyase by running 2 volumes of Heparin Lyasethrough the column.

After allowing 60 minutes for enzymatic cleavage of the glycosidelinkage, the unbound complexes of Vaccinia virus particles andheparin-residues bound to the Vaccinia virus particles are eluted withPBS, 0.02 M phosphate, 0.15 M NaCl, pH 7.5. The Vaccinia virusparticle-GAG-residue complex is dissociated with PBS 0.02 M, 0.3 M NaCl,pH 7.5. The Heparin-residues are removed by diafiltration.

The eluate is analyzed by titration for viable (infectious) Vacciniavirus particles by a Tissue Culture cytopatic effect assay (TCID50), fortotal number of Vaccinia virus particles by a real-time qPCR forVaccinia DNA, for host cell protein by use of the BCA total proteinassay and for host cell DNA by use of a real-time qPCR. The recovery canbe >70% and biological activity of the recovered Vaccinia virus can be>80%.

Example 7

Two ml of a highly concentrated and previously purified Vaccinia viruspreparation with approximately 2×10⁹ virus particles per ml were appliedto a Sulfated Reinforced Cellulose membrane.

The membrane was washed with PBS 0.01 M, 0.15 M NaCl, pH 7.5. The A280absorbance signal was used for monitoring of Vaccinia virus particle andthe host cell protein concentrations until it returned to baseline (thepre-loading value). The washings were continued for a total of 25minutes.

The bound Vaccinia virus particles were eluted by a NaCl concentrationgradient in PBS 0.01 M, pH 7.5. The concentration of NaCl was increasedlinearly from 0.15 M to 2.0 M. The elution started after approximately atotal of 30 minutes (5 minutes after starting the gradient). The majorpeak was eluted 5 minutes later (at T=35 minutes). The peak contained ahigh concentration of Vaccinia virus particles as assessed by the LaserScattering signal used for monitoring of Vaccinia virus particles.

The eluate was analyzed by a Vaccinia Virus specific ELISA showing avirus recovery rate of approx 40%. Host-cell protein was analyzed by useof the BCA total protein assay and showed a protein recovery of approx5% in the eluate. Host-cell DNA was analyzed by a total DNA assay andshowed approx 5% DNA in the eluate.

Example 8

Production of Modified Vaccinia Ankara Virus Particles

MVA-BN® virus particles were produced in primary cultures of CEF cellsunder Good Manufacturing Practice conditions. The starting material forthis study was provided after homogenization and clarification as aliquid frozen product, stored in aliquots at −20° C. or −80° C.

MVA-BN®-Quantification

Virus titers were determined in triplicates by a sandwich ELISA. Therelative virus amounts were correlated to the initial TCID₅₀ value. Ascapturing antibody a rabbit anti-Vaccinia virus (Cat.# 220100717;Quartett Immunodiagnostika & Biotechnologie GmbH, Germany) was used. Thedetection antibody was a peroxidase conjugated polyclonal rabbitanti-Vaccinia virus antibody (Cat.# 8104; ViroStat, USA).

Total Protein Assay

Total protein concentrations were determined in triplicates by thePierce® BCA protein assay reagent kit (Cat.# 23225; PierceBiotechnology, USA) according to the manufacture's instructions. Theassay was calibrated against albumin standards (BSA) (Cat.# 23209;Thermo Fisher Scientific Inc., USA) within the validated working rangeof 25 to 250 μg/ml (LOD: 8.3 μg/ml; LOQ: 25 μg/ml) using 100 mM citricacid and 250 mM NaCl buffer pH 7.2 for dilutions. All samples wereadjusted to the same buffer conditions.

dsDNA-assay

The dsDNA measurements were done as described by Opitz et al. (Opitz etal., Vaccine 25(5):939-947, 2007) using the Quant-iT™ PicoGreen® dsDNAreagent from Molecular Probes, Inc. (Cat.# P7581, USA). The assay wascalibrated against lambda DNA (Cat.# D1501, Promega Corporation, USA)within the validated working range of 4 to 1000 ng/ml (weightedregression; LOD: 0.66 ng/ml; LOQ: 2.36 ng/ml) using 100 mM citric acidbuffer pH 7.2 for dilutions. The same buffer was used for samplepreparation via dialysis (5000 kDa MWCO; Cat.# 131192, Spectrum EuropeB.V., Netherlands) and sample dilutions. After incubation of standardsand samples with the reagent, the fluorescent signal was measured at anemission and excitation wavelength of 535 nm and 485 nm, respectively(Mithras LB 940, Berthold Technologies GmbH & Co.KG, Germany). Allsamples were measured in duplicates.

Chromatography Materials

Pseudo-affinity membrane adsorbers—Heparin-MA (Sartorius Stedim BiotechGmbH, Germany) was based on reinforced stabilized cellulose with a poresize >3 μm and an adsorption area of 3×75 cm² by 3×15 layers. Thehousing material was polypropylene. Sulfated cellulose MA (SC-MA) with adiameter of 25 mm (pore size >3 μm, Sartorius Stedim Biotech GmbH,Germany) were prepared as described previously (Opitz et al.,Biotechnol. and Bioeng. 103(6):1144-1154, 2009). The adsorption area was75 cm² and 15 membranes were stacked in a stainless steel membraneholder (Cat.# 1980-002, GE Healthcare, Germany).

Ion exchange membrane adsorbers—Dynamic binding capacity studies andcapturing experiments were conducted by four different ion exchange MA:A strong anion exchange MA (Sartobind Q75-MA; Cat.# Q75X; 75 cm², 15layers), a weak anion exchange MA (Sartobind D75-MA; Cat.# D75X; 75 cm²,15 layers), a strong cation exchange MA (Sartobind S75-MA; Cat.# S75X;75 cm², 15 layers) and a weak cation exchange MA (Sartobind 075-MA;Cat.# C75X; 75 cm², 15 layers). All ion exchange MA were from SartoriusStedim Biotech GmbH, Germany.

Bead-based pseudo-affinity resins—Cellufine® sulfate (3 ml, Cat. #19845,Chisso Corporation, Japan), and Toyopearl AF-Heparin HC-650M (3 ml, Cat.# 20030, Tosoh Bioscience, Germany) were packed into a Tricorn 5/150column (GE Healthcare, Germany).

Adsorption Chromatography

Chromatography was performed using an Akta Explorer system (GEHealthcare, Germany) at a flow rate of 0.5 ml/min (unless stateddifferently) and monitored by UV (280 nm) and light scattering (90°,Dawn EOS, Wyatt Technology Inc., USA) detection.

Dynamic binding capacity of the chromatography media was determinedloading the clarified MVA-BN® virus sample (4.65×10⁷ TCID₅₀/ml) at aflow rate of 0.5 ml/min onto Sartobind S75-MA, C75-MA, Q75-MA, D75-MA,Heparin-MA and SC-MA. All applied MA had a surface area of 75 cm² andwere composed of 15 layers. In parallel, the dynamic binding capacitywas determined for the 3 ml Cellufine® sulfate and Toyopearl® AF-Heparinbeads. The breakthrough was monitored via light scattering detector.

Characterization of the chromatography materials was done with 4 ml ofthe clarified MVA-BN® virus sample, representing a dynamic bindingcapacity of approximately 49% for the C75-MA (3×75 cm²), 22% for theheparin-MA (3×75 cm²) and less than 20% for the Q75-MA (75 cm²), D75-MA(75 cm²), SC-MA (75 cm²) and the 3 ml Cellufine® sulfate column. Priorto sample loading, the chromatography material was equilibrated withsample buffer (100 mM citric acid, pH 7.2). After a brief washing theadsorbed virus particles were eluted with elution buffer (100 mM citricacid, 2 M NaCl, pH 7.2). Resulting fractions were pooled and analyzedfor virus and contaminant compositions. Chromatographic materials wereregenerated after each run with 10 column volumes of 1 M NaOH and 0.1 MHCl in 1 M NaCl. Dynamic binding studies were performed in triplicatesfor Cellufine® sulfate and the heparin-MA and once for all othermaterials (Table 2). All other experiments were performed at least intriplicates, the precise number of experiments is indicated in FIG. 1A.

TABLE 2 Dynamic binding capacity of the tested chromatography materials.Breakthrough Chromatography Volume Total TCID₅₀ Media Functional GroupsN (ml) (TCID₅₀) Q75-MA Quaternary ammonium 3 >20 >9.3 × 10⁸  D75-MADiethylamine 3 >20 >9.3 × 10⁸  S75-MA Sulfonic acid 3 2.5 1.2 × 10⁸C75-MA Carboxyl 3 2.7 1.3 × 10⁸ Heparin-MA Heparin 3 6.0 2.8 × 10⁸ SC-MASulfated cellulose 3 >20 >9.3 × 10⁸  (~20 μg/g dry membrane) Toyopearl ®AF- Heparin 3 3.0 1.4 × 10⁸ Heparin (3 ml) Cellufine ® Sulfatedcellulose 3 >20 >9.3 × 10⁸  sulfate (3 ml) (≧700 μg/g dry gel) Theadsorption area of all membrane adsorbers was 75 cm². The applied bufferwas 100 mM citric acid (pH 7.2).

Combination of Membrane Adsorbers

The chromatography was performed using the same system and monitored asdescribed above. All membrane adsorbers were equilibrated with samplebuffer (100 mM citric acid, pH 7.2) before virus adsorption. Six ml ofthe clarified MVA-BN® virus sample were subjected to a SC-MA (75 cm²) ora heparin-MA (225 cm²) at a flow rate of 10 ml/min. After a briefwashing (sample buffer, 10 ml/min) the adsorbed virus particles wereeluted (100 mM citric acid, 2 M NaCl, pH 7.2; 0.5 ml/min), pooled anddialysed against sample buffer with a MWCO of 5000 kDa (Cat.# 131192,Spectrum Europe B.V., Netherlands). Dialysed samples were furtherpurified via a Q75-MA (75 cm²; FIG. 2) applying identical operatingconditions as for the pseudo-affinity MA, except for the adsorption flowrate of 0.5 ml/min.

The virus content and the amount of total dsDNA and protein weredetermined from a representative sample as described above. Analyticalsamples removed were considered in the overall mass balances.

Separation of MVA-BN® by Membrane Adsorption Chromatography andCellufine® Sulfate Column Chromatography

Both tested anion exchange MA (Q75-MA and D75-MA) had a dynamic bindingcapacity of >20 ml culture broth capturing MVA-BN® virus particles(Table 2). Comparable dynamic binding capacities (>20 ml) were achievedby the sulfated cellulose based pseudo-affinity matrices (SC-MA and 3 mlCellufine® sulfate column). A reduced capacity of 6.0 ml and 3.0 ml wasobserved for the heparin matrices, the heparin-MA and the 3.0 mlToyopearl AF-Heparin column, respectively. The tested cation exchangeMA, S75-MA and C75-MA, resulted in a dynamic binding capacity of 2.5 mland 2.7 ml, respectively.

Due to the low dynamic binding capacity of the 3 ml Toyopearl®AF-Heparin column and the S75-MA these matrices were not furthercharacterized. Studies with the C-75-MA were continued despite the lowdynamic binding capacity to compare the performance of a weak cationexchanger with the negatively charged pseudo-affinity MA. To increasethe capacity of the C75-MA and heparin-MA, three 75 cm² units wereserially combined to obtain an overall adsorption area of 225 cm².

Viruses have been shown to bind sulfated polysaccharides such as dextransulfate, heparin, and heparan sulphate (Lycke et al., J Gen Virol72(5):1131-1137, 1991; Mitsuya et al. 1988; O'Keeffe et al.,Biotechnology and Bioengineering 62(5):537-545, 1999; O'Neil et al.,1993; Opitz et al. 2009). Here, a significant difference in the dynamicbinding capacities for the tested heparin and sulfated cellulosematrices was observed. However, the heparin density, the averagemolecular weight of the heparin ligands as well as their degree and typeof sulfation are not provided from the respective manufacturers. Theseparameters can play a role for the heparin target interaction (Feyzi etal., J. Biol. Chem. 272(40):24850-24857, 1997; Marks et al., Journal ofMedicinal Chemistry 44(13):2178-2187, 2001; Rusnati et al., J. Biol.Chem. 272(17):11313-11320 1997).

Further investigations of the ion exchange MA for the capturing ofMVA-BN® virus particles revealed that a fraction of MVA-BN® virusparticles (21%; FIG. 1A) adsorbed to the weak cation exchanger C75-MAand was desorbed by an increased ionic strength via NaCl. A similarresult, even more pronounced, was described by Opitz et al. forpurification of cell culture-derived influenza virus particles (Opitz etal. 2009). The overall pl of influenza virus particles as judged by thesubtype A₂/Singapore/57 (5.0; Zhilinskaya et al., Acta Virol.16(5):436-439, 1972) is less acidic than the pl of Vaccinia virusparticles. Nevertheless, based on their overall pl both types of virusesare not expected to bind at neutral buffer conditions to cationexchangers. However, the surface charge of larger particles (i.e.virions) influences significantly their adsorption behaviour. This mightexplain the adsorption characteristics of MVA-BN® and influenza virusparticles to cation exchange matrices.

The virus content in the product fraction of the Q75-MA and D75-MA was77% and 72% of the initial MVA-BN® virus (FIG. 1A). Fourteen to 17% ofthe virus particles did not adsorb to the matrices. In addition, forboth anion exchange MA the overall virus balance could not be closed anda small portion of the initial virus particles could not be accountedfor. Those particles could presumably not be desorbed during the elutionbut only during the following regeneration. Incomplete desorption ofvirus particles was also described for cell culture-derived influenzavirus particles from two anion exchange MA (i.e. Q75-MA and D75-MA)(Kalbfuss et al. 2007), supporting the observations described here.These losses might be due to strong adsorption of virus subfractions orvirus debris, which are accounted for in the analytics. Alternatively,the losses may be explained by a filtration effect of MA, which isparticularly relevant in the case of large virus aggregates. However, ifthe appropriate membrane pore size has been selected for the virusparticles, product losses due to filtration effects are minimal as canbe judged from high virus recoveries observed by Opitz et al. after aSC-MA capture of influenza virus particles (Opitz et al. 2009).Furthermore, unspecific product losses have also been described in theliterature for proteins during membrane chromatography (Sorci et al.,Desalination 199(1-3):550-552, 2006). Hence, the observed losses ofMVA-BN® virus particles are not necessarily related to the particlesize.

One striking drawback of anion exchange MA was the co-adsorption ofdsDNA. Vaccinia viruses have a double-stranded DNA genome (Antoine etal., Virology 244(2):365-396, 1998). Hence differentiation between hostcell DNA and virus DNA cannot be accomplished by the applied total DNAassay. However, after host cell homogenization it is evident, that onlya minor fraction of the measured total dsDNA is viral DNA. Pooledproduct fractions of the Q75-MA and D75-MA contained 84% and 70% dsDNA,respectively (FIG. 1B). Kalbfuss et al. established a NaCl step gradientfor a bead-based anion exchanger, which allowed the separation ofinfluenza virus particles from the contaminating host cell DNA duringthe desorption process (Kalbfuss et al., Biotechnology andBioengineering 96(5):932-944, 2007). This was not possible for MVA-BN®virus particles and the host cell DNA of primary CEF cells with theapplied MA (data not shown). Hence, the product fraction was heavilycontaminated with dsDNA (FIG. 1B). However, some of the adsorbed dsDNAwas not desorbed during the elution process. This dsDNA was eitherdesorbed during the regeneration or bound irreversibly to the MA,allowing a remote reduction of dsDNA in the product fraction. Similarstudies on the purification of plasmid DNA via anion exchange membranecapsules also reported partial dsDNA losses (Syren et al. 2007). In thecase of the cation exchange MA (C75-MA) dsDNA amounted to 26% in theproduct fraction (FIG. 1). However, virus particles also adsorbed poorlyto the C75-MA, providing no possibility for an efficient separation ofMVA-BN® virus particles from host cell DNA.

The advantage of an affinity adsorption process is its high specificityallowing an improved purity and a good product yield at optimaloperating conditions and ligand selections. Vaccinia virus produces fourdifferent types of progeny virus from infected cells: intracellularmature virus (IMV), intracellular enveloped virus (IEV), cell-associatedenveloped virus (CEV) and extracellular enveloped virus (EEV). Thesurface proteins differ between IMV and the other progeny virusparticles. So far glycosaminoglycans have only been reported to bind tosurface proteins of IMV (Ho et al. 2005; Hsiao et al. 1999; Lin et al.2000; Resch et al., Virology 358(1):233-47, 2007; Smith, G. L. 2002, J.Gen. Virol. 83: 2915-2931). Hence, selecting heparin and sulfatedcellulose matrices may lead to reduced virus yields. However, IMV is themost abundant form of the infectious progeny (Hsiao et al. 1999; Reschet al., Smith, 2002) and therefore the most prominent target for anaffinity chromatography. Furthermore, it has to be considered that IMV,compared to the extracellular progeny, remains within the cell untillysis (Smith et al. 2002), pointing out the importance of cellhomogenisation to achieve optimal virus yields. The possible selectionof IMV via heparin and sulfated cellulose ligands might be reflected inthe amount of virus which did not adsorb to the SC-MA (30%) andheparin-MA (35%; FIG. 1A). The average amount of MVA-BN® in the productfractions of the SC-MA was slightly higher with 65% compared to theheparin-MA with 56% (FIG. 1A). As before, the material balances couldnot be closed completely. A small fraction of the virus particlesremained on the matrix and was most likely desorbed during theregeneration process. Furthermore, the bead-based Cellufine® sulfate ledto comparable results for the overall virus yields (59%). Differencesbetween the two MA and Cellufine® sulfate were observed in the depletionof dsDNA. The Cellufine® sulfate product fraction included 7% of thestarting amount of dsDNA. The values for the heparin-MA and CS-MA were24% and 10%, respectively (FIG. 1B). Hence, use of the heparin-MAresulted in at least 2 times the amount of contaminating dsDNA than useof the sulfated cellulose matrices.

The relative amounts of total protein, based on the loaded sample, whichdid not adsorb to the tested matrices ranged from 86% (heparin-MA) to102% (Cellufine® sulfate) and was in general slightly higher for the ionexchange MA (Q75-MA (92%), D75-MA (93%) and C75-MA (98%)) as compared tothe pseudo-affinity MA (heparin-MA (86%) and SC-MA (88%); FIG. 1C).However, the quantity of total protein in the product fractions wasbelow 1% in all tested chromatography materials (FIG. 1C). Hence, asobserved for the virus particles and the dsDNA a small fraction of theloaded protein interacted with the tested matrices too strong to bedesorbed during the elution process. Similar observations have beendescribed by Opitz et al. during the purification of influenza virusparticles (Opitz et al., Journal of Biotechnology 131(3):309-317, 2007).

Although virus recovery was slightly higher with ion exchange MA, thelow level of dsDNA contamination of pseudo-affinity matrices clearlyindicates a superior performance of these adsorbers to capture CEFcell-derived MVA-BN® virus particles. Furthermore, the limited dynamicbinding capacities of the tested cation exchangers (S75-MA (1.5 ml) andC75-MA (2.0 ml); Tab. 1) in comparison to the sulfated cellulosematrices (Cellufine® sulfate (>20 ml) and the SC-MA (>20 ml)) are astrong indication that the interaction between the MVA-BN® particles andthe SC-MA is not solely based on its negative charge. Similarobservations have been reported by Opitz et al., which describedsignificant differences for the depletion of host cell DNA from cellculture-derived influenza virus particles comparing SC-MA with weak andstrong cation exchange MA (Opitz et al. 2009). On the other hand, virusyields were in their studies comparable between the SC-MA and the strongcation exchange membrane adsorber S75-MA, while virus recovery for theweak cation exchange membrane adsorber C75-MA was slightly reduced(Opitz et al. 2009). However, the improved adsorption of influenza virusparticles to cation exchange resins in comparison to MVA-BN® can beexplained by the reduced acidity of the influenza virus and theiraccessible surface charge. Hence, the observed differences in dsDNAdepletion from Opitz et al. and the significant differences in the virusadsorption for cation exchange MA and the SC-MA described here supportthe conclusion that the interaction between MVA-BN® virus particles andsulfated cellulose was not only determined by the ionic charge of thematrix.

The main advantage of MA compared to conventional column chromatographyfor purification of large components (i.e. virus particles) is thereduced pressure drop, allowing operations at higher flow rates. In thecase of affinity MA processes, the flow rate is mainly limited by theassociation and dissociation kinetics of the ligand-target complex. Asshown in Table 3, an increase in flow rate from 0.5 ml/min to 10 ml/minresulted in a decrease in virus recovery for the SC-MA (14%) and theheparin-MA (12%) with a total virus recovery in the product fraction of51% and 47%, respectively. The amount of dsDNA in the product fractionat 10 ml/min was slightly reduced compared to 0.5 ml/min for bothadsorbers (Tab. 2). The recommended operating pressure (manufacture'sinstruction) for Cellufine® sulfate is less than 2 bar. For the appliedset-up, as described in the material and methods section, this limitedthe flow rate to 0.5 ml/min for bead-based separations. A change incolumn dimensions would have allowed an increase in the flow rate forCellufine® sulfate. However, wide column dimensions at a constant matrixvolume lead to a reduced residence time, resulting in potential productlosses. On the other hand, up-scaling of the complete matrix volume hasto be questioned in terms of the process economics.

TABLE 3 Effect of increased flow rates on viral recovery and contaminantdepletion. Flow Rate MVA-BN ® dsDNA Total Protein (ml/min) MembraneAdsorber (%) (%) (%) 0.5 Cellufine ® sulfate 59 ± 5.7 7.0 ± 1.9 0.2 ±0.2 0.5 SC-MA 65 ± 0.5 6.0 ± 0.2 <LOQ^(a) 5.0 SC-MA 54 ± 0.3 4.0 ± 1.0<LOQ^(a) 10.0 SC-MA 51 ± 0.2 4.0 ± 0.7 <LOQ^(a) 0.5 Heparin-MA 59 ± 1.3 17 ± 2.1 <LOQ^(a) 5.0 Heparin-MA 47 ± 3.9  11 ± 0.3 <LOQ^(a) 10.0Heparin-MA 47 ± 0.4   9 ± 3.1 <LOQ^(a) Relative amounts (mean andstandard deviation of triplicates) for MVA-BN ® (ELISA), dsDNA and totalprotein content were calculated based on the starting material of thehomogenized and clarified virus broth. The adsorption area of the SC-MAand heparin-MA was 75 cm² and 225 cm², respectively. Equilibration andwash buffer was 100 mM citric acid (pH 7.2), and the elution buffer 100mM citric acid + 2M NaCl (pH 7.2). ^(a)limit of quantification (25μg/ml)

The overall performance based on capacity, purity, virus yield andproductivity of the SC-MA was significantly better compared to ionexchange MA and heparin-MA. In terms of productivity, the SC-MA canclearly be favoured over the bead-based Cellufine® sulfate as it ispossible to increase the flow rates 20-fold for the SC-MAchromatography. Furthermore, the study demonstrated that the interactionof MVA-BN® particles to sulfated cellulose is most likely not only ofelectrostatic nature. In fact, it has to be a complex interactioncomparable to other biological affinity chromatography systems.Certainly, it should be considered that the selection of heparin andsulfated cellulose as pseudo-affinity matrix to capture MVA-BN® virusparticles potentially leads to the exclusion of certain virus progenies.However, the development of a specific but virus progeny independentaffinity ligand might not be justified by the low virus losses.Actually, it might be more promising to optimize the ligand density ofheparin or sulfated cellulose MA.

Combination of Pseudo-Affinity and Anion Exchange Membrane Adsorbers

Regulatory expectations of host cell DNA contents in CEF-cell producedlicensed vaccines are less stringent than current regulatoryrequirements for new vaccine products. Current guidelines for newlylicensed human vaccine products from continuous cell lines stipulatethat residual DNA levels exceeding 10 ng per dose are not acceptable(European-Pharmacopoeia 2009 (Version 6.4); World-Health-Organization1998). As expected, these requirements for a new vaccine product can notbe accomplished by a single initial capture step. Lowest levels fordsDNA in product fractions using MA were obtained with the SC-MA andheparin-MA. In a subsequent process step the remaining dsDNA could beeliminated by a nuclease treatment, which is routinely done viaBenzonase® treatment for smallpox vaccines (Greenberg and Kennedy 2008;Monath et al. 2004) and other vaccine preparations like influenzavaccines (Wolff and Reichl, Chemical Engineering & Technology31(6):846-857, 2008). Currently, for smallpox vaccines the Benzonase®treatments are mainly carried out after homogenization and clearance(Greenberg and Kennedy 2008; Monath et al. 2004). An application of theBenzonase® treatment in the purification scheme after thepseudo-affinity MA would significantly reduce total process costs due tothe reduction of the total amount of Benzonase® required. As analternative, the host cell DNA content could be further reduced byintroduction of an additional unit operation, i.e. an anion exchange MA.Therefore, sequential capturing and purification of the MVA-BN® virusparticles was explored as the next step (FIG. 2). As a result, theamount of dsDNA in the product fraction was reduced to 0.1% and 5%respectively. The overall virus recovery for the SC-MA and heparin-MAset-up was 58% and 59%, respectively. Moreover, after introduction ofthe subsequent anion exchange MA protein contaminations in the productfractions were reduced below the quantification limit (5 μg/ml) for bothpurification schemes.

On the other hand, differential elution of MVA-BN® particles and dsDNAfrom the Q75-MA was not possible. Both components co-eluted during awide range of salt-concentrations (data not shown), but part of thedsDNA was desorbed during the regeneration process or bound irreversiblyto the anion exchange MA (FIG. 1B). This could be exploited for afurther reduction of the dsDNA content in the product fraction. Virusyields of the Q75-MA after both pseudo-affinity chromatography methodsexceeded the yields obtained from loading the homogenized MVA-BN®particles directly to the Q75-MA. Virus losses were in both cases onlyabout 4% (FIG. 2) while the dsDNA was reduced, relative to the startingmaterial, from 4% to 0.1% for the SC-MA scheme and for the heparin-MAscheme from 23% to 5%. Other MA or purification methods could be used tofurther deplete the dsDNA to comply with requirements for human vaccineproducts. However, even if the necessary limits for host cell DNA arereached, it should be considered that a Benzonase® treatment alsoreduces the probability of vaccines to contain intact oncogenes or otherfunctional DNA sequences in vaccines. (Knezevic et al., Biologicals36(3):203-211 2008).

Pseudo-affinity MA allowed the capture of CEF cell-derived MVA-BN® at ahigh loading velocity (10 ml/min) with a relatively high purity.Compared to the bead-based pseudo-affinity matrix Cellufine® sulfate(0.5 ml/min), productivity could be increased by a factor of 20 with aslightly reduced product yield. The achieved purity levels, inparticular the dsDNA depletion, were significantly higher for thepseudo-affinity matrices than for the tested ion exchange MA. Forproduction of new vaccines products or virus vectors further dsDNAreduction is required and an improved viral yield would still bedesirable. Preliminary experiments with SC-MA, which have been sulfatedby a modified chemical reaction, indicated the potential to improvevirus yields significantly. Overall, the pseudo-affinity MA represent avaluable choice to capture Vaccinia virus particles in a manufacturingprocess and potentially allow to economise the required Benzonase®treatment step compared to classical downstream processes for smallpoxvaccines.

TABLE 1 VV surface proteins Surface protein (gene) VV form ReferencesA2.5L MV  [1] A9L MV  [2] A13L MV  [3] A14L MV [4-7] A14.5L MV  [8] A16LMV  [9; 10] A17L MV [11-13] A21L MV [14] A25L MV [15] A26L MV [15; 16]A27L MV [17-22] A28L MV [23; 24] A33R EV [25-27] A34 R EV [28-31] A36RWV [32-37] A38L [38; 39] A56R EV [40-43] B5R EV [44-46] D8L MV [47] D13LMV E1OR MV [48] F9L MV [49] F12 WV [37; 50] F13L EV [51-55] G3L MV  [9]G4L MV [56; 57] G9R MV  [9; 58] H2 R MV [59] H3L MV [60-62]  12L MV [63]I5L MV [64] J5L MV  [9] K2L WV/EV [65-68] L1R MV [69] L5R MV [70] Listof References cited in Table 1: [1] Senkevich T G, White C L, WeisbergA, Granek J A, Wolfe E J, Koonin E V, Moss B: Expression of the vacciniavirus A2.5L redox protein is required for virion morphogenesis. Virology2002; 300(2): 296-303. [2] Yeh W W, Moss B, Wolfe E J: The vacciniavirus A9L gene encodes a membrane protein required for an early step invirion morphogenesis. J Virol 2000; 74(20): 9701-9711. [3] Unger B,Traktman P: Vaccinia virus morphogenesis: a13 phosphoprotein is requiredfor assembly of mature virions. J Virol 2004; 78(16): 8885-8901. [4]Traktman P, Liu K, DeMasi J, Rollins R, Jesty S, Unger B: Elucidatingthe essential role of the A14 phosphoprotein in vaccinia virusmorphogenesis: construction and characterization of atetracycline-inducible recombinant. J Virol 2000; 74(8): 3682-3695. [5]Rodriguez J R, Risco C, Carrascosa J L, Esteban M, Rodriguez D: Vacciniavirus 15-kilodalton (A14L) protein is essential for assembly andattachment of viral crescents to virosomes. J Virol 1998; 72(2):1287-1296. [6] Mercer J, Traktman P: Investigation of structural andfunctional motifs within the vaccinia virus A14 phosphoprotein, anessential component of the virion membrane. J Virol 2003; 77(16):8857-8871. [7] Betakova T, Wolffe E J, Moss B: Regulation of vacciniavirus morphogenesis: phosphorylation of the A14L and A17L membraneproteins and C-terminal truncation of the AI 7L protein are dependent onthe F1OL kinase. J Virol 1999; 73(5): 3534-3543. [8] Betakova T, WolffeE J, Moss B: The vaccinia virus A14.5L gene encodes a hydrophobic53-amino-acid virion membrane protein that enhances virulence in miceand is conserved among vertebrate poxviruses. J Virol 2000; 74(9):4085-4092. [9] Senkevich T G, Ojeda S, Townsley A, Nelson G E, Moss B:Poxvirus multiprotein entry-fusion complex. Proc Natl Acad Sci USA 2005;102(51): 18572-18577. [10] Ojeda S, Senkevich T G, Moss B: Entry ofvaccinia virus and cell-cell fusion require a highly conservedcysteine-rich membrane protein encoded by the AI 6L gene. J Virol 2006;80(1): 51-61. [11] Betakova T, Wolfe E J, Moss B: Membrane topology ofthe vaccinia virus A17L envelope protein. Virology 1999; 261(2):347-356. [12] Rodriguez D, Esteban M, Rodriguez J R: Vaccinia virus A17Lgene product is essential for an early step in virion morphogenesis. JVirol 1995; 69(8): 4640-4648. [13] Wolffe E J, Moore D M, Peters P J,Moss B: Vaccinia virus A17L open reading frame encodes an essentialcomponent of nascent viral membranes that is required to initiatemorphogenesis. J Virol 1996; 70(5): 2797-2808. [14] Townsley A C,Senkevich T G, Moss B: Vaccinia virus A21 virion membrane protein isrequired for cell entry and fusion. J Virol 2005; 79(15): 9458-9469.[15] Ulaeto D, Grosenbach D, Hruby D E: The vaccinia virus 4c and A-typeinclusion proteins are specific markers for the intracellular maturevirus particle. J Virol 1996; 70(6): 3372-3377. [16] McKelvey T A,Andrews S C, Miller S E, Ray C A, Pickup D J: Identification of theorthopoxvirus p4c gene, which encodes a structural protein that directsintracellular mature virus particles into A-type inclusions. J Virol2002; 76(22): 11216-11225. [17] Ho Y, Hsiao J C, Yang M H, Chung C S,Peng Y C, Lin T H, Chang W, Tzou D L: The oligomeric structure ofvaccinia viral envelope protein A27L is essential for binding to heparinand heparan sulfates on cell surfaces: a structural and functionalapproach using site-specific mutagenesis. J Mol Biol 2005; 349(5):1060-1071. [18] Vazquez M I, Esteban M: Identification of functionaldomains in the 14- kilodalton envelope protein (A27L) of vaccinia virus.J Virol 1999; 73(11): 9098-9109. [19] Hsiao J C, Chung C S, Chang W:Cell surface proteoglycans are necessary for A27L protein-mediated cellfusion: identification of the N-terminal region of A27L protein as theglycosaminoglycan-binding domain. J Virol 1998; 72(10): 8374-8379. [20]Chung C S, Hsiao J C, Chang Y S, Chang W: A27L protein mediates vacciniavirus interaction with cell surface heparan sulfate. J Virol 1998;72(2): 1577-1585. [21] Vazquez M I, Rivas G, Cregut D, Serrano L,Esteban M: The vaccinia virus 14-kilodalton (A27L) fusion protein formsa triple coiled-coil structure and interacts with the 21-kilodalton(A17L) virus membrane protein through a C-terminal alpha-helix. J Virol1998; 72(12): 10126-10137. [22] Rodriguez D, Rodriguez J R, Esteban M:The vaccinia virus 14-kilodalton fusion protein forms a stable complexwith the processed protein encoded by the vaccinia virus AI 7L gene. JVirol 1993; 67(6): 3435-3440. [23] Senkevich T G, Ward B M, Moss B:Vaccinia virus entry into cells is dependent on a virion surface proteinencoded by the A28L gene. J Virol 2004; 78(5): 2357-2366. [24] SenkevichT G, Ward B M, Moss B: Vaccinia virus A28L gene encodes an essentialprotein component of the virion membrane with intramolecular disulfidebonds formed by the viral cytoplasmic redox pathway. J Virol 2004;78(5): 2348-2356. [25] Roper R L, Wolffe E J, Weisberg A, Moss B: Theenvelope protein encoded by the A33R gene is required for formation ofactin-containing microvilli and efficient cell-to-cell spread ofvaccinia virus. J Virol 1998; 72(5): 4192-4204. [26] Roper R L, Payne LG, Moss B: Extracellular vaccinia virus envelope glycoprotein encoded bythe A33R gene. J Virol 1996; 70(6): 3753-3762. [27] Wolffe E J, WeisbergA S, Moss B: The vaccinia virus A33R protein provides a chaperonefunction for viral membrane localization and tyrosine phosphorylation ofthe A36R protein. J Virol 2001; 75(1): 303-310. [28] Duncan S A, Smith GL: Identification and characterization of an extracellular envelopeglycoprotein affecting vaccinia virus egress. J Virol 1992; 66(3):1610-1621. [29] Rottger S, Frischknecht F, Reckmann I, Smith G L, Way M:Interactions between vaccinia virus IEV membrane proteins and theirroles in IEV assembly and actin tail formation. J Virol 1999; 73(4):2863-2875. [30] Wolffe E J, Katz E, Weisberg A, Moss B: The A34Rglycoprotein gene is required for induction of specializedactin-containing microvilli and efficient cell-to-cell transmission ofvaccinia virus. J Virol 1997; 71(5): 3904-3915. [31] McIntosh A A, SmithG L: Vaccinia virus glycoprotein A34R is required for infectivity ofextracellular enveloped virus. J Virol 1996; 70(1): 272-281. [32]Sanderson C M, Frischknecht F, Way M, Hollinshead M, Smith G L: Roles ofvaccinia virus EEV-specific proteins in intracellular actin tailformation and low pH-induced cell-cell fusion. J Gen Virol 1998; 79 (Pt6): 1415-1425. [33] Scaplehorn N, Holmstrom A, Moreau V, Frischknecht F,Reckmann I, Way M: Grb2 and Nck act cooperatively to promote actin-basedmotility of vaccinia virus. Curr Biol 2002; 12(9): 740-745. [34] van EH, Hollinshead M, Smith G L: The vaccinia virus A36R protein is a typeIb membrane protein present on intracellular but not extracellularenveloped virus particles. Virology 2000; 271(1): 26-36. [35] Ward B M,Moss B: Vaccinia virus A36R membrane protein provides a direct linkbetween intracellular enveloped virions and the microtubule motorkinesin. J Virol 2004; 78(5): 2486-2493. [36] Wolfe E J, Weisberg A S,Moss B: Role for the vaccinia virus A36R outer envelope protein in theformation of virus-tipped actin-containing microvilli and cell-to-cellvirus spread. Virology 1998; 244(1): 20-26. [37] van E H, Hollinshead M,Rodger G, Zhang W H, Smith G L: The vaccinia virus F12L protein isassociated with intracellular enveloped virus particles and is requiredfor their egress to the cell surface. J Gen Virol 2002; 83: 195-207.[38] Parkinson J E, Sanderson C M, Smith G L: The vaccinia virus A38Lgene product is a 33-kDa integral membrane glycoprotein. Virology 1995;214(1): 177-188. [39] Sanderson C M, Parkinson J E, Hollinshead M, SmithG L: Overexpression of the vaccinia virus A38L integral membrane proteinpromotes Ca2+ influx into infected cells. J Virol 1996; 70(2): 905-914.[40] Seki M, Oie M, Ichihashi Y, Shida H: Hemadsorption and fusioninhibition activities of hemagglutinin analyzed by vaccinia virusmutants. Virology 1990; 175(2): 372-384. [41] Shida H: Nucleotidesequence of the vaccinia virus hemagglutinin gene. Virology 1986;150(2): 451-462. [42] Shida H, Dales S: Biogenesis of vaccinia:carbohydrate of the hemagglutinin molecules. Virology 1981; 111(1):56-72. [43] Payne L G, Norrby E: Presence of haemagglutinin in theenvelope of extracellular vaccinia virus particles. J Gen Virol 1976;32(1): 63-72. [44] Katz E, Wolfe E J, Moss B: The cytoplasmic andtransmembrane domains of the vaccinia virus B5R protein target achimeric human immunodeficiency virus type 1 glycoprotein to the outerenvelope of nascent vaccinia virions. J Virol 1997; 71(4): 3178-3187.[45] Herrera E, Lorenzo M M, Blasco R, Isaacs S N: Functional analysisof vaccinia virus B5R protein: essential role in virus envelopment isindependent of a large portion of the extracellular domain. J Virol1998; 72(1): 294-302. [46] Ward B M, Moss B: Golgi network targeting andplasma membrane internalization signals in vaccinia virus B5R envelopeprotein. J Virol 2000; 74(8): 3771-3780. [47] Hsiao J C, Chung C S,Chang W: Vaccinia virus envelope D8L protein binds to cell surfacechondroitin sulfate and mediates the adsorption of intracellular maturevirions to cells. J Virol 1999; 73(10): 8750-8761. [48] Senkevich T G,Weisberg A S, Moss B: Vaccinia virus E1OR protein is associated with themembranes of intracellular mature virions and has a role inmorphogenesis. Virology 2000; 278(1): 244-252. [49] Senkevich T G, WhiteC L, Koonin E V, Moss B: A viral member of the ERV1/ALR protein familyparticipates in a cytoplasmic pathway of disulfide bond formation. ProcNatl Acad Sci USA 2000; 97(22): 12068-12073. [50] Zhang W H, Wilcock D,Smith G L: Vaccinia virus F12L protein is required for actin tailformation, normal plaque size, and virulence. J Virol 2000; 74(24):11654-11662. [51] Blasco R, Moss B: Extracellular vaccinia virusformation and cell-to-cell virus transmission are prevented by deletionof the gene encoding the 37,000-Dalton outer envelope protein. J Virol1991; 65(11): 5910-5920. [52] Husain M, Moss B: Similarities in theinduction of post-Golgi vesicles by the vaccinia virus F13L protein andphospholipase D. J Virol 2002; 76(15): 7777-7789. [53] Husain M,Weisberg A, Moss B: Topology of epitope-tagged F13L protein, a majormembrane component of extracellular vaccinia virions. Virology 2003;308(2): 233-242. [54] Roper R L, Moss B: Envelope formation is blockedby mutation of a sequence related to the HKD phospholipid metabolismmotif in the vaccinia virus F13L protein. J Virol 1999; 73(2):1108-1117. [55] Schmutz C, Rindisbacher L, Galmiche M C, Wittek R:Biochemical analysis of the major vaccinia virus envelope antigen.Virology 1995; 213(1): 19-27. [56] White C L, Senkevich T G, Moss B:Vaccinia virus G4L glutaredoxin is an essential intermediate of acytoplasmic disulfide bond pathway required for virion assembly. J Virol2002; 76(2): 467-472. [57] White C L, Weisberg A S, Moss B: Aglutaredoxin, encoded by the G4L gene of vaccinia virus, is essentialfor virion morphogenesis. J Virol 2000; 74(19): 9175-9183. [58] Ojeda S,Domi A, Moss B: Vaccinia virus G9 protein is an essential component ofthe poxvirus entry-fusion complex. J Virol 2006; 80(19): 9822-9830. [59]Senkevich T G, Moss B: Vaccinia virus H2 protein is an essentialcomponent of a complex involved in virus entry and cell-cell fusion. JVirol 2005; 79(8): 4744-4754. [60] da Fonseca F G, Wolffe E J, WeisbergA, Moss B: Effects of deletion or stringent repression of the H3Lenvelope gene on vaccinia virus replication. J Virol 2000; 74(16):7518-7528. [61] da Fonseca F G, Wolffe E J, Weisberg A, Moss B:Characterization of the vaccinia virus H3L envelope protein: topologyand posttranslational membrane insertion via the C-terminal hydrophobictail. J Virol 2000; 74(16): 7508-7517. [62] Lin C L, Chung C S, Heine HG, Chang W: Vaccinia virus envelope H3L protein binds to cell surfaceheparan sulfate and is important for intracellular mature virionmorphogenesis and virus infection in vitro and in vivo. J Virol 2000;74(7): 3353-3365. [63] Knipe D M, Howley P M: Fields Virology, ed Fifth,Philadelphia, USA, Lippincott Williams & Wilkins, a Wolthers Kluwerbusiness, 2007. [64] Takahashi T, Oie M, Ichihashi Y: N-terminal aminoacid sequences of vaccinia virus structural proteins. Virology 1994;202(2): 844-852. [65] Law K M, Smith G L: A vaccinia serine proteaseinhibitor which prevents virus-induced cell fusion. J Gen Virol 1992; 73(Pt 3): 549-557. [66] Turner P C, Moyer R W: An orthopoxvirus serpinlikegene controls the ability of infected cells to fuse. J Virol 1992;66(4): 2076-2085. [67] Turner P C, Moyer R W: The cowpox virus fusionregulator proteins SPI-3 and hemagglutinin interact in infected anduninfected cells. Virology 2006; 347(1): 88-99. [68] Zhou J, Sun X Y,Fernando G J, Frazer I H: The vaccinia virus K2L gene encodes a serineprotease inhibitor which inhibits cell-cell fusion. Virology 1992;189(2): 678-686. [69] Su H P, Garman S C, Allison T J, Fogg C, Moss B,Garboczi D N: The 1.51-Angstrom structure of the poxvirus L1 protein, atarget of potent neutralizing antibodies. Proc Natl Acad Sci USA 2005;102(12): 4240-4245. [70] Townsley A C, Senkevich T G, Moss B: Theproduct of the vaccinia virus L5R gene is a fourth membrane proteinencoded by all poxviruses that is required for cell entry and cell-cellfusion. J Virol 2005; 79(17): 10988-10998.

We claim:
 1. An industrial-scale method for the purification ofbiologically active Vaccinia viruses comprising: i) loading asolid-phase matrix comprising a sulfated cellulose with biologicallyactive Vaccinia viruses, wherein the solid-phase matrix comprises or isa membrane; ii) washing the matrix; and iii) eluting a minimum of5.0×10¹² Vaccinia virus particles.
 2. The method of claim 1, wherein theVaccinia viruses are recombinant Vaccinia viruses.
 3. The method ofclaim 1, wherein the Vaccinia viruses are modified Vaccinia Ankaraviruses or recombinant modified Vaccinia Ankara viruses.
 4. The methodof claim 1, wherein the solid-phase matrix comprises a membrane.
 5. Themethod of claim 1, wherein the solid-phase matrix is a membrane.
 6. Themethod of claim 4, wherein the solid-phase matrix comprises a sulfatedreinforced cellulose membrane.
 7. The method of claim 4, wherein thesolid-phase matrix is a sulfated reinforced cellulose membrane.
 8. Themethod of claim 1, wherein the Vaccinia viruses are eluted with sodiumchloride (NaCl).
 9. The method of claim 1, additionally comprising apurification step by ion-exchange.
 10. The method of claim 9, whereinthe purification step by ion-exchange comprises a membrane.
 11. Themethod of claim 1, further comprising administering the eluted Vacciniaviruses to an animal.
 12. The method of claim 11, wherein the animal isa human.
 13. An aseptic method for the purification of biologicallyactive Vaccinia virus comprising: i) aseptically loading a solid-phasematrix comprising a sulfated cellulose with a biologically activeVaccinia virus, wherein the solid-phase matrix comprises or is amembrane; ii) aseptically washing the matrix; and iii) asepticallyeluting the biologically active Vaccinia virus.
 14. The method of claim13, wherein the Vaccinia virus is a recombinant Vaccinia virus.
 15. Themethod of claim 13, wherein the Vaccinia virus is a modified VacciniaAnkara virus or a recombinant modified Vaccinia Ankara virus.
 16. Themethod of claim 13, wherein the solid-phase matrix comprises a membrane.17. The method of claim 13, wherein the solid-phase matrix is amembrane.
 18. The method of claim 16, wherein the solid-phase matrixcomprises a sulfated reinforced cellulose membrane.
 19. The method ofclaim 16, wherein the solid-phase matrix is a sulfated reinforcedcellulose membrane.
 20. The method of claim 13, wherein the Vacciniavirus is eluted with sodium chloride (NaCl).
 21. The method of claim 13,additionally comprising a purification step by ion-exchange.
 22. Themethod of claim 21, wherein the purification step by ion-exchangecomprises a membrane.
 23. The method of claim 13, further comprisingadministering the eluted Vaccinia virus to an animal.
 24. The method ofclaim 23, wherein the animal is a human.